Cardiomyocytes From Induced Pluripotent Stem Cells From Patients and Methods of Use Thereof

ABSTRACT

Human somatic cells obtained from individuals with a genetic heart condition are reprogrammed to become induced pluripotent stem cells (iPS cells), and differentiated into cardiomyocytes for use in analysis, screening programs, and the like.

CROSS-REFERENCE

This application claims benefit and is a Continuation of Application ofSer. No. 13/554,946 filed Jul. 20, 2012, which claims benefit of U.S.Provisional Patent Application No. 61/510,422, filed Jul. 21, 2011,which applications are incorporated herein by reference in theirentirety.

FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with Government support under contract HL099776awarded by the National Institutes of Health. The Government has certainrights in the invention.

BACKGROUND OF THE INVENTION

A variety of cardiac disorders have an underlying genetic cause. Forexample, dilated cardiomyopathy (DCM) is a cardiac disease characterizedby ventricular dilatation and systolic dysfunction. DCM is the mostcommon cause of heart failure after coronary artery disease andhypertension, as well as the leading indication for hearttransplantations. The cost for management of DCM in the US alone hasbeen estimated at between $4 and $10 billion. Another importantcondition for therapy is hypertrophic cardiomyopathy (HCM), in which thesarcomeres replicate, causing cardiomyocytes to increase in size. Inaddition, the normal alignment of cardiomyocytes is disrupted, aphenomenon known as myocardial disarray. HCM is most commonly due to amutation in one of nine sarcomeric genes.

Mutations in genes encoding sarcomeric, cytoskeletal, mitochondrial, andnuclear membrane proteins, as well as proteins involved in calciummetabolism, are associated with approximately a third to half the casesof DCM. Cardiac troponin T (cTnT) is one of the 3 subunits of thetroponin complex (Troponin T, C, and I) that regulate the sarcomericthin filament activity and muscle contraction in cardiomyocytes (CMs).cTnT is essential for sarcomere assembly, contraction, and forceproduction. Mutations in the cardiac troponin T gene (TNNT2) often leadto DCM and are frequently expressed as a malignant phenotype with suddencardiac death and heart failure at an early age. In vitro biochemicalstudies have found that decreased Ca²⁺ sensitivity and/or ATPaseactivity, which lead to impaired force production, may be the underlyingmechanisms for certain TNNT2-mutation induced DCM.

Mouse models of TNNT2 mutations recapitulate the human DCM phenotype andhave provided extensive insight into the possible mechanisms of thedisease. However, several differences exist between the mouse and humanmodels. For example, mouse resting heart rate is approximately 10-foldfaster than human. The electrical properties, ion channel contributions,and cardiac development of mouse CMs are also different from those ofhuman. The lack of complex intracellular interactions withincardiomyocytes for in vitro biochemical assays and species differencesfor mouse models undercut the value of these methodologies forunderstanding the cellular and physiological processes of DCM as well asfor drug screening.

In addition, cardiac tissues from DCM patients are difficult to obtainand do not survive in long-term culture. Effective cellular models fordilated cardiomyopathy and other genetic cardiac conditions are of greatinterest for screening and development of effective therapies.

Pharmaceutical drug discovery, a multi-billion dollar industry, involvesthe identification and validation of therapeutic targets, as well as theidentification and optimization of lead compounds. The explosion innumbers of potential new targets and chemical entities resulting fromgenomics and combinatorial chemistry approaches over the past few yearshas placed enormous pressure on screening programs. The rewards foridentification of a useful drug are enormous, but the percentages ofhits from any screening program are generally very low. Desirablecompound screening methods solve this problem by both allowing for ahigh throughput so that many individual compounds can be tested; and byproviding biologically relevant information so that there is a goodcorrelation between the information generated by the screening assay andthe pharmaceutical effectiveness of the compound.

Some of the more important features for pharmaceutical effectiveness arespecificity for the targeted cell or disease, a lack of toxicity atrelevant dosages, and specific activity of the compound against itsmolecular target. The present invention addresses this issue.

PUBLICATIONS

Methods to reprogram primate differentiated somatic cells to apluripotent state include differentiated somatic cell nuclear transfer,differentiated somatic cell fusion with pluripotent stem cells anddirect reprogramming to produce induced pluripotent stem cells (iPScells) (Takahashi K, et al. (2007) Cell 131:861-872; Park I H, et al.(2008) Nature 451:141-146; Yu J, et al. (2007) Science 318:1917-1920;Kim D, et al. (2009) Cell Stem Cell 4:472-476; Soldner F, et al. (2009)Cell. 136:964-977; Huangfu D, et al. (2008) Nature Biotechnology26:1269-1275; Li W, et al. (2009) Cell Stem Cell 4:16-19).

Additional publications of interest include Stadtfeld et al. Science322, 945-949 (2008); Okita et al. Science 322, 949-953 (2008); Kaji etal. Nature 458, 771-775 (2009); Soldner et al. Cell 136, 964-977 (2009);Woltjen et al. Nature 458, 766-770 (2009); Yu et al. Science (2009).

SUMMARY OF THE INVENTION

Compositions and methods are provided for disease-relevant screening ofcardiomyocytes for therapeutic drugs and treatment regimens, where themethods utilize in vitro cell cultures or animal models derivedtherefrom. Diseases of particular interest include dilatedcardiomyopathy (DCM); hypertrophic cardiomyopathy (HCM);anthracycline-induced cardiotoxicity; arrhythmogenic right ventriculardysplasia (ARVD); left ventricular non-compaction (LVNC); double inletleft ventricle (DILV); and long QT (Type-1) syndrome (LQT-1), in whichthere is a genetic basis for the disease. The methods utilize inducedhuman pluripotent stem cells (iPS cells), which may be obtained frompatient or carrier cell samples, e.g. adipocytes, fibroblasts, and thelike.

In some embodiments of the invention, in vitro cell cultures ofdisease-relevant cardiomyocytes are provided, where the cardiomyocytesare differentiated from induced human pluripotent stem cells (iPS cells)comprising at least one allele encoding a mutation associated with acardiac disease. Mutations of interest include mutations in the genes:cardiac troponin T (TNNT2); myosin heavy chain (MYH7); tropomyosin 1(TPM1); myosin binding protein C (MYBPC3); 5′-AMP-activated proteinkinase subunit gamma-2 (PRKAG2); troponin I type 3 (TNNI3); titin (TTN);myosin, light chain 2 (MYL2); actin, alpha cardiac muscle 1 (ACTC1);potassium voltage-gated channel, KQT-like subfamily, member 1 (KCNQ1);plakophilin 2 (PKP2); and cardiac LIM protein (CSRP3). Specificmutations of interest include, without limitation, MYH7 R663H mutation;TNNT2 R173W; PKP2 2013delC mutation; PKP2 Q617X mutation; and KCNQ1G269S missense mutation.

In some embodiments a panel of such cardiomyocytes are provided, wherethe panel includes two or more different disease-relevantcardiomyocytes. In some embodiments a panel of such cardiomyocytes areprovided, where the cardiomyocytes are subjected to a plurality ofcandidate agents, or a plurality of doses of a candidate agent.Candidate agents include small molecules, i.e. drugs, genetic constructsthat increase or decrease expression of an RNA of interest, electricalchanges, and the like. In some embodiments the disease-relevantcardiomyocytes are introduced or induced to differentiate from iPS cellsin an in vivo environment, for example as an explant in an animal model.In some embodiments a panel refers to a system or method utilizingpatient-specific cardiomyocytes from two or more distinct cardiacconditions, and may be three or more, four or more, five or more, six ormore, seven or more distinct conditions, where the conditions areselected from: dilated cardiomyopathy (DCM); hypertrophic cardiomyopathy(HCM); anthracycline-induced cardiotoxicity; arrhythmogenic rightventricular dysplasia (ARVD); left ventricular non-compaction (LVNC);double inlet left ventricle (DILV); and long QT (Type-1) syndrome(LQT-1).

In some embodiments of the invention, methods are provided fordetermining the activity of a candidate agent on a disease-relevantcardiomyocyte, the method comprising contacting the candidate agent withone or a panel of cardiomyocytes differentiated from induced humanpluripotent stem cells (iPS cells) comprising at least one alleleencoding a mutation associated with a cardiac disease; and determiningthe effect of the agent on morphologic, genetic or functionalparameters, including without limitation calcium transient amplitude,intracellular Ca²⁺ level, cell size contractile force production,beating rates, sarcomeric α-actinin distribution, and gene expressionprofiling. Methods of analysis at the single cell level are ofparticular interest, e.g. atomic force microscopy, microelectrode arrayrecordings, patch clamping, single cell PCR, calcium imaging, and thelike.

Where the disease is DCM, the cardiomyocytes may be stimulated withpositive inotropic stress, such as a β-adrenergic agonist before, duringor after contacting with the candidate agent. In some embodiments theβ-adrenergic agonist is norepinephrine. It is shown herein that DMCcardiomyocytes have an initially positive chronotropic effect inresponse to positive inotropic stress, that later becomes negative withcharacteristics of failure such as reduced beating rates, compromisedcontraction, and significantly more cells with abnormal sarcomericα-actinin distribution. β-adrenergic blocker treatment andover-expression of sarcoplasmic reticulum Ca²⁺ ATPase (Serca2a) improvethe function. DCM cardiomyocytes may also be tested with genetic agentsin the pathways including factors promoting cardiogenesis, integrin andcytoskeletal signaling, and ubiquitination pathway, for example as shownin Table 8. Compared to the control healthy individuals in the samefamily cohort, DCM cardiomyocytes exhibit decreased calcium transientamplitude, decreased contractility, and abnormal sarcomeric α-actinindistribution.

Where the disease is HCM the cardiomyocytes may be stimulated withpositive inotropic stress, such as a β-adrenergic agonist before, duringor after contacting with the candidate agent. Under such conditions, HCMcardiomyocytes display higher hypertrophic responses, which can bereversed by a β-adrenergic blocker. Compared to healthy individuals, HCMcardiomyocytes exhibit increased cell size and up-regulation of HCMrelated genes, and more irregularity in contractions characterized byimmature beats, including a higher frequency of abnormal Ca²⁺transients, characterized by secondary immature transients. Thesecardiomyocytes have increased intracullar Ca²⁺ levels, and in someembodiments candidate agents that target calcineurin or other targetsassociated with calcium affinity.

Also provided are pluripotent stem cell populations comprising at leastone allele encoding a mutation associated with a cardiac disease.Mutations of interest include mutations in the genes: cardiac troponin T(TNNT2); myosin heavy chain (MYH7); tropomyosin 1 (TPM1); myosin bindingprotein C (MYBPC3); 5′-AMP-activated protein kinase subunit gamma-2(PRKAG2); troponin I type 3 (TNNI3); titin (TTN); myosin, light chain 2(MYL2); actin, alpha cardiac muscle 1 (ACTC1); potassium voltage-gatedchannel, KQT-like subfamily, member 1 (KCNQ1); plakophilin 2 (PKP2); andcardiac LIM protein (CSRP3). Specific mutations of interest include,without limitation, MYH7 R663H mutation; TNNT2 R173W; PKP2 2013delCmutation; PKP2 Q617X mutation; and KCNQ1 G269S missense mutation. Thepluripotent stem cell populations may be provided as a cell culture,optionally a feeder-layer free cell culture. Various somatic cells finduse as a source of iPS cells; of particular interest are adipose-derivedstem cells, fibroblasts, and the like. The pluripotent cells andcardiomyocytes derived therefrom may be used for transplantation, forexperimental evaluation, as a source of lineage and cell specificproducts, and the like. These and other objects, advantages, andfeatures of the invention will become apparent to those persons skilledin the art upon reading the details of the subject methods andcompositions as more fully described below.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is best understood from the following detailed descriptionwhen read in conjunction with the accompanying drawings. The patent orapplication file contains at least one drawing executed in color. Copiesof this patent or patent application publication with color drawing(s)will be provided by the Office upon request and payment of the necessaryfee. It is emphasized that, according to common practice, the variousfeatures of the drawings are not to-scale. On the contrary, thedimensions of the various features are arbitrarily expanded or reducedfor clarity. Included in the drawings are the following figures.

FIG. 1. Generation of patient-specific DCM iPSCs. (a) Schematic pedigreeof the seven member DCM family recruited in this study. Filled squares(male) and circles (female) represent individuals carrying the specificTNNT2 R173W mutation on chromosome 1 in one of the two alleles. (b) TheR173W point mutation was confirmed to be present on exon 12 of the TNNT2gene in the DCM patients by PCR and DNA sequencing. CON, control. (c) Arepresentative image of the patient-derived skin fibroblasts expandedfrom the skin biopsies. Representative images of an (d) ESC-like and (e)TRA-1-60 positive colony derived from reprogramming the patient-derivedskin fibroblasts with Yamanaka factors. (f) Immunofluorescence andalkaline phosphatase staining of the patient skin fibroblasts-derivediPSCs. (g) Quantitative bisulphite pyrosequencing analysis of themethylation status at the promoter regions of Oct4 and Nanog in thepatient-specific iPSCs. Both the Nanog and Oct4 promoter regions werehighly demethylated in the patient-specific iPSCs. (h) Teratomas derivedfrom the patient-specific iPSCs injected into the kidney capsule ofimmunodeficient mice showing tissues of all three embryonic germ layers.Bars, 200 μm.

FIG. 2. DCM iPSC-CMs exhibited a significant higher number of cells withabnormal sarcomeric α-actinin distribution and early failure after NEtreatment. (a) Immunostaining of sarcomeric α-actinin and cTnT at day 30post differentiation. Single DCM iPSC-CMs exhibited punctate sarcomericα-actinin distribution pattern suggesting a disorganized myofilamentstructure. Enlarged views of the boxed areas of the merged micrographsshowing detailed α-actinin and cTnT staining pattern in the cells. Bars,20 μm. (b) Compared to control iPSC-CMs (n=368), a significant higherpercentage of DCM iPSC-CMs (n=391) showed punctate sarcomeric α-actininstaining pattern in greater than one fourth of the total cellular area(**p=0.008). (c) No significant difference was observed in cell sizebetween control (n=36) and DCM iPSC-CMs (n=39). (d) A representative MEAassay tracking contraction properties of both control (n=14) and DCM(n=14) beating EBs overtime with or without NE treatment. EBs wereseeded in a dual chamber MEA probe with one side treated with NE and theother without. Electrical signals were recorded simultaneously duringthe experiments. Beating frequencies were normalized to those of EBswithout NE treatment. (e) Normalized beating frequencies of CM clusters(n=10) over time after NE treatment by video imaging. (f) Representativeimages of sarcomeric α-actinin immunostaining on single control and DCMiPSC-CMs after 7 days of NE treatment. Compared to the controls, longterm NE treatment significantly aggravated sarcomeric organization ofDCM cells. Bar, 20 μm. (g) Percentage of CMs with disorganizedsarcomeric staining pattern with (control, n=210; DCM, n=255) or without(control, n=261; DCM, n=277) NE treatment. NE treatment markedlyincreased the number of disorganized CMs in DCM group (**p<0.001), andhad a less significant effect on control iPSC-CMs (*p=0.05). (h)Tracking morphological and contractility changes of iPSC-CMs overtimeafter NE treatment. Bar, 200 μm. Data are presented as mean±s.e.m.

FIG. 3. DCM iPSC-CMs exhibited smaller [Ca²⁺]_(i) transients. (a)Representative line-scan images and (b) spontaneous calcium transientsin CON (left) and DCM iPSC-CMs (right). (c) Frequency of spontaneouscalcium transients in control and DCM iPSC-CMs. (d) Integration of[Ca²⁺]_(i) transients in control and DCM iPSC-CMs showed less Ca²⁺released in each transient in DCM relative to control cells (control,n=87 cells; DCM, n=40, **P=0.002). There were no significant differencesin the (e) irregularity of timing (standard deviation/mean) or (f)amplitude of the spontaneous calcium transients between CON and DCMcells.

FIG. 4. Over-expression of Serca2a restored contractility of DCMiPSC-CMs. (a), Western blotting of Serca2a expression after adenoviraltransduction of DCM iPSC-CMs. Serca2a protein level was upregulated incells transduced with Ad.Serca2a but not in cells transduced withAd.GFP. (b) A representative image showing the AFM cantileverapproaching GFP positive single beating CMs. Bar, 50 μm. (c) Histogramsof contraction forces of all the single iPSC-CMs measured by AFM over100-400 beats. Over-expression of Serca2a significantly restored thecontraction force of DCM iPSC-CMs to a level close to that of thecontrols. (d) Dot plots of mean contraction force of single CMs measuredby AFM. One-way ANOVA analysis indicated that there was significantdifference among the mean of all the groups (**p=0.002). Tukey'sMultiple Comparison Test indicated that both control iPSC-CMs (n=13)(P=0.001) and Ad.Serca2a (n=12) (P=0.005) transduced DCM iPSC-CMsexhibited significantly stronger contraction force than that transducedby Ad.GFP (n=17). Ad.Serca2a transduced DCM iPSC-CMs showed comparablecontraction force to that of the control iPSC-CMs (p=0.578). (e)Representative spontaneous calcium transients in single DCM iPSC-CMstransduced with Ad.GFP and Ad.Serca2a, respectively. (f) DCM iPSC-CMstransduced with Ad.Serca2a (n=22) exhibited increased global calciumtransients compared to cells transduced with Ad.GFP (n=14). (*p=0.04)(two-tailed Student's t-test). (g) Percentage of CMs with disorganizedsarcomeric staining pattern in DCM iPSC-CMs with Ad.Serca2a (n=40) orAd.GFP (n=40) over-expression. No significant difference was observedbetween the two groups (two-tailed Student's t-test). Data are presentedas mean±s.e.m.

FIG. 5. R173W mutation in the iPSCs derived from DCM patients in thefamily. Genomic PCR of the locus of TNNT2 and DNA sequencing indicatethat iPSCs from all DCM patients carried the R173W (C to T) mutation.

FIG. 6. Comparison of the global mRNA expression patterns of human ESCs(H7), skin fibroblasts, and patient-specific iPSCs by microarray. BothPearson correlation and scatter plots indicate that the global geneexpression pattern of patient-specific iPSCs was highly similar to thatof human ESCs.

FIG. 7. Patient-specific iPSCs maintained normal karyotype afterextended culture. Representative images from two DCM iPSC lines wereshown after culturing for 20 passages.

FIG. 8. Quantitative-PCR of relative expression levels of total versusendogenous Yamanaka reprogramming factors. By comparing the total andendogenous gene expression level of each reprogramming factor, exogenoustransgenes Oct4, Sox2, Klf4, and c-MYC were silenced in most of theestablished patient-specific iPSCs. Endogenous Nanog expression wasup-regulated in all the patient-specific iPSCs, indicating apluripotency state of each cell line. Note that the Nanog expressionlevels were normalized to that of the H7 human ESCs (not shown). Primerinformation used for quantitative PCR are listed in Supplementary Table6.

FIG. 9. Patient-specific iPSCs can differentiate into cells from the 3germ layers in vitro. Different cell types, such as neurons, endothelialcells, red blood cells, as well as cells expressing mesoderm markersmooth muscle actin (SMA), endoderm marker α-fetoprotein (AFP), andectoderm marker (Tuj-1) were detected from spontaneous differentiationof all the patient-specific iPSCs. Bars, 100 μm.

FIG. 10. Relative cardiac differentiation efficiency of thepatient-specific iPSCs. The cardiac differentiation efficiency isrepresented as percentage of beating EBs (n=3 for each line, data arepresented as mean±s.e.m).

FIG. 11. (a) and (b) FACS analysis of percentage of cTnT positive CMswithin beating EBs derived from control and DCM iPSCs. ˜50-60% cells inbeating EBs were cTnT positive cardiomyocytes.

FIG. 12. Allele-specific PCR of wild type (Wt) and mutant (R173W) TNNT2expression in DCM and control iPSC-CMs. Patient IIa, IIb, and IIIa wereconfirmed to express the mutant TNNT2 in their respective iPSC-derivedCMs. The primers used for allelic PCR are listed in Supplementary Table6.

FIG. 13. Multi-electrode arrays (MEA) examining electrophysiologicproperties of iPSC-derived beating EBs. (a) The MEA probe and arepresentative image of 4 beating EBs seeded. (b) The electrical signalsrecorded by MEA reflecting field potentials of the 4 beating EBs shownin (a). (c) Extracted MEA field potential graphs showing field potentialduration (FPD), maximum positive amplitude (MPA), maximum negativeamplitude (MNA), and interspike interval (ISI).

FIG. 14. Single cell PCR analyzing gene expression levels in 24 controland 24 DCM iPSC-CMs at day 30 post differentiation. Gene expression of(a) cardiac specific transcription factors, (b) calcium handling relatedproteins, (c) ion channels, (d) sarcomeric proteins, and (e) skeletalmuscle specific proteins relative to gene expression level of α-tubulinwere analyzed. No significant differences were observed between controland DCM CMs. Data are presented as mean±s.e.m. Statistical differencewas tested using two tailed Student's T-test.

FIG. 15. iPSC-CMs expressed cardiac-specific proteins. Both control andDCM iPSC-CMs expressed cardiac specific proteins sarcomeric α-actinin,cTnT, connexin43, and MLC2a. Arrows indicate positive connexin43staining at the cell-cell contact. Bars, 20 μm.

FIG. 16. Enlarged view of immunostaining of sarcomeric α-actinin andcTnT in the single CMs shown in FIG. 2a . Bar, 20 μm.

FIG. 17. Double immunostaining of sarcomeric α-actinin and cTnT in thesingle CMs shown in FIG. 2f . Bar, 20 μm.

FIG. 18. Enlarged view of merged graph of each cell shown in FIG. 2f .Note that after NE treatment, some single DCM iPSC CMs showed completedegeneration of myofilaments, which was not observed in control CMs.Bar, 20 μm.

FIG. 19. (a-c) Real time PCR on single DCM iPSC-CMs versus controliPSC-CMs showed gene expression changes after one week of NE treatment.Control and DCM iPSC-CMs were seeded on culture dishes at day 19 postdifferentiation and were treated with or without 10 μM NE 48 h later for7 days. Single CMs from control (treated with NE, n=8; without NE, n=8)and DCM (treated with NE, n=8; without NE, n=8) iPSCs were picked andPCR were performed as described in the Method section. Net thresholdcycle (CT) values between cells treated with NE and without NE werefirst calculated. Data were then presented as the net CT values of theDCM group relative to the net ct values of control group. Genes weregrouped as upregulated (>1 cycle difference in CT), downregulated (>1cycle difference in CT), and no expression changes (<1 cycle differencein CT) after NE treatment.

FIG. 20. Electrophysiological features of iPSC-CMs measured by patchclamping. (a) Three types of spontaneous AP were observed in bothcontrol and DCM iPSC-CMs (left, ventricular-like; center, atrial-like;right, nodal-like). An estimated 70-80% cells were ventricular-like CMs,whereas the others were atrial- and/or nodal-like cells. There is nosignificant difference in cardiac cell fate between control and DCMiPSCs (data not shown). (b) Spontaneous AP in control and DCMventricular myocytes using current-clamp recording. DCM ventricularcells had slightly shorter APs compared to control cells (P=0.112) (c).There was no significant difference in the frequency (d), the peakamplitude of AP (e), or in the resting membrane potential (f) betweencontrol and DCM cells at the time of measurements (day 19-day 25 postdifferentiation) (control, n=18; DCM, n=17). Statistical difference wastested using the two tailed Student's T-test.

FIG. 21. Atomic force microscopy (AFM) measurement of contraction forceof iPSC-CMs. (a) Schematic of the process of force measurement by AFM ata single cardiomyocyte level. (b) A representative image showing AFMcantilever probing a single cardiomyocyte. Bar, 20 μm. (c) Arepresentative graph showing the signals acquired by AFM and theparameters examined (force, frequency, and beat duration).

FIG. 22. Beat frequency and duration of single iPSC-CMs measured by AFM.(a) Dot plots of mean beat frequency measured by AFM. No significantdifference in beat frequency and rhythm was observed between controliPSC-CMs (n=13), Ad.Serca2a (n=12) and Ad.GFP (n=17) transduced DCMiPSC-CMs. (b) Dot plots of mean beat duration measured by AFM.Over-expression of Serca2a significantly shortened the beat duration(*p=0.029). Statistical difference was tested using one-way ANOVAfollowed by Tukey's Multiple Comparison Test. (c) Histograms of beatfrequency and (d) beat duration of all the single iPSC-CMs measured byAFM over 100-400 beats.

FIG. 23. Dot plots of relative cell size versus contraction force foreach single cell measured by AFM. There is no significant linearrelationship between cell size and contraction force in (a) control(R²=0.006), (b) DCM/DCM-Ad.GFP (R²=0.105), and (c) DCM-Ad.Serca2a(R²=0.061) groups.

FIG. 24. Normalized percentage of beating foci in culture dish over timeafter Serca2a and GFP over-expression. Data represent averages of threeindependent replicates of experiments (mean±s.e.m.).

FIG. 25. Ca²⁺ imaging of iPSC-CMs transduced with Ad.Serca2a or Ad.GFPadenoviruses with red fluorescent Ca²⁺ indicator Rhod-2 AM. (a) Mergedconfocal images showing the GFP positive CMs uptook the Rhod-2 dye. (b)The same cell in (a) was scanned with the arrow line indicated in thepicture. (c) The line scan images recorded for the particular CM shownin (a) and (b). Bar, 20 μm.

FIG. 26. Contractility of control iPSC-CMs transduced with Ad.Serca2a orAd.GFP as measured by AFM. (a) Dot plots of contraction force, (b) beatfrequency, and (c) beat duration of control iPSC-CMs transduced withAd.Serca2a (n=10) or Ad.GFP (n=10). No significant statisticaldifferences were observed between the mean of each group. Statisticaldifference was tested using two tailed Student's T-test. (d) Histogramsof contraction force, (e) beat frequency, and (f) beat duration of allthe single control iPSC-CMs measured by AFM over 100-400 beats.

FIG. 27. Gene expression profiling of DCM iPSC-CMs with Serca2aover-expression identified enriched pathways that may function inrescuing the DCM phenotype. (a) Heatmap of the 191 genes with greaterthan 1.5-fold difference in expression in biological replicates ofcontrol iPSC-CMs and Serca2a-treated DCM iPSC-CMs compared with DCMiPSC-CMs without Serca2a treatment. (b) Heatmap of enriched pathwaysthat may be involved in rescuing the DCM phenotype by Serca2aover-expression.

FIG. 28. Metoprolol treatment improved sarcomeric organization of DCMiPSC-CMs and alleviate the aggravation effect of NE treatment. (a) TenμM metoprolol treatment increased the number of DCM iPSC-CMs with intactsarcomeric integrity (untreated, n=100; treated, n=86, *p=0.023). (b)Metoprolol treatment prevented the aggravation of DCM iPSC-CMs inducedby NE treatment. Both 1 μM (n=107, **p=0.008) and 10 μM (n=101,**p=0.001) metoprolol significantly decreased the number of disorganizedcells compared to those without metoprolol treatment (n=108). (c) Ten μMmetoprolol treatment had no significant effect on the sarcomericintegrity of control iPSC-CMs (untreated, n=88; treated, n=75). Data arepresented as mean±s.e.m. Statistical difference was tested using the twotailed Student's T-test.

FIG. 29. Similar functional properties of DCM iPSC-ECs and controliPSC-ECs. (a) FACS analyses indicated the efficiency of differentiationof both DCM and control iPSCs to CD31⁺ ECs were similar. (b) The FACSisolated CD31⁺ cells from differentiated DCM and control iPSCs expressedboth the endothelial cell markers CD31 and CD144. (c) Both DCM andcontrol iPSC-derived ECs exhibited uptake capability of low densitylipoprotein (LDL) (red fluorescence). (d) Both control and DCMiPSC-derived ECs were able to form web-like tubules on Matrigel surface.Bars, 100 μm.

FIG. 30. Schematic of potential mechanisms by Serca2a gene therapy inDCM iPSC-CM. The mutation in cardiac troponin T negatively affectscontractility, sarcomere formation, and calcium signaling, which causeschanges in calcium-related genes such as calsequestrin, NFAT, and TRICchannels. However, electrical excitation was normal in the TNNT2 R173WDCM iPSC-CMs. Delivery of Serca2a, the SR/ER membrane calcium pump,restored the level of calcium handling molecules, reversed thecompromised calcium transients and contractility, and thereby improvedthe overall DCM iPSC-CM function. cTnT, cardiac troponin T; LTC, L-typecalcium channel; RyR, ryanodine receptor calcium release channel; CSQ,calsequestrin; PM, plasma membrane.

FIG. 31. Generation and characterization of patient-specific HCMiPSC-CMs. (A) Representative long-axis MRI images of the proband and acontrol matched family member at end systole and end diastoledemonstrating asymmetric hypertrophy of the inferior wall. (B)Confirmation of the Arg663His missense mutation on exon 18 of the MYH7gene in HCM patients (II-1, III-1, III-2, III-3, and III-8) by PCR andsequence analysis. (C) Schematic pedigree of the proband carrying theArg663His mutation in MYH7 recruited for this study (II-1) as well asher husband (II-2), and eight children (III-1 through III-8). Circlesrepresent female family members and squares represent males. Solidsymbols indicate clinical presentation of the HCM phenotype, whereasopen symbols represent absence of presentation. “+” and “−” signsunderneath family members indicate presence and absence of the Arg663Hismutation respectively. Two individuals (III-3 and III-8) were found tocarry the Arg663His mutation but had yet to present the HCM phenotypedue to young age. (D) Representative immunostaining for cardiac troponinT and F-actin demonstrating increased cellular size and multinucleationin HCM iPSC-CMs as compared to control iPSC-CMs. (E) Quantification ofcell size for 4 control iPSCCM lines (II-2, III-4, III-6, III-7) (n=55per patient line) and 4 HCM iPSC-CM lines (II-1, III-1, III-2, III-8)(n=59 per patient line) 40 days after induction of cardiacdifferentiation. (F) Quantification of multi-nucleation in control(n=55, 4 patient lines) and HCM iPSC-CMs (n=59, 4 patient lines). (G)Representative immunofluorescence staining reveals elevated ANFexpression in HCM iPSC-CMs as compared to controls. (H) Changes in ANFgene expression as measured by single cell quantitative PCR in controland HCM iPSC-CMs at days 20, 30, and 40 following induction of cardiacdifferentiation (n=32 per time point, 5 patient lines). (I)Quantification of MYH7/MYH6 expression ratio in HCM iPSC-CMs andcontrols (n=32 per time point, 5 patient lines). (J) Representativeimmunofluorescence staining images revealing nuclear translocation ofNFATC4 in HCM iPSC-CMs. (K) Percentage of cardiomyocytes exhibitingpositive NFATC4 staining in control (n=187, 5 patient lines) and HCMiPSC-CMs (n=169, 5 patient lines). (L) Quantification of cell size incontrol and HCM iPSC-CMs following treatment with calcineurin inhibitorsCs-A and FK506 for 5 continuous days (n=50, 5 patient lines per group).(M) Heat map representations of gene expression in single control andHCM iPSC-CMs for genes associated with cardiac hypertrophy at days 20,30, and 40 following induction of cardiac differentiation. * denotesP<0.05 HCM vs control, ** denotes P<0.0001 HCM vs control.

FIG. 32. Assessment of arrhythmia and irregular Ca²⁺ regulation in HCMiPSC-CMs. (A) Electrophysiological measurements of spontaneous actionpotentials in control and HCM iPSCCMs measured by patch clamp incurrent-clamp mode. Boxes indicate underlined portions of HCM iPSC-CMwaveforms at expanded timescales demonstrating DAD-like arrhythmias. (B)Quantification of DAD occurrence in control (n=144, 5 patient lines) andHCM iPSC-CMs (n=131, 5 patient lines). DAD rate is defined as totalDADs/total beats. (C) Quantification of percentage of control (n=144, 5patient lines) and HCM iPSC-CMs (n=131, 5 patient lines) exhibitingputative DADs. (D) Representative line-scan images and spontaneous Ca²⁺transients in control and HCM iPSC-CMs. Red arrows indicatetachyarrhythmia-like waveforms observed in HCM cells but not control.(E) Quantification of percentages for control and HCM iPSC-CMsexhibiting irregular Ca²⁺ transients at days 20, 30, and 40 followinginduction of cardiac differentiation (n=50, 5 patient lines pertimepoint). (F) Representative line-scan images and spontaneous Ca²⁺transients for H9 hESC-CMs and hESC-CMs stably transduced withlentivirus driving expression of wild type MYH7 or mutant MYH7 carryingthe Arg663His mutation. Red arrowheads indicate irregular Ca²⁺waveforms. (G) Quantification of cells exhibiting irregular Ca²⁺transients in WA09 hESC-CMs, hESC-CMs overexpressing wild-type MYH7, andhESCCMs overexpressing MYH7 carrying the Arg663His mutation (n=40, 5patient lines per group). (H) Spontaneous action potentials recorded incurrent-clamp mode for hESC-CMs, hESC-CMs overexpressing wild type MYH7,and hESC-CMs overexpressing MYH7 carrying the Arg663His mutation. Redarrowheads indicate DAD-like waveforms. (I) Quantification of cellsexhibiting DAD-like waveforms in hESC-CMs, hESC-CMs stably transducedwith lentivirus driving expression of wild type MYH7 or mutant MYH7carrying the Arg663His mutation (n=20, 5 patient lines per group). (J)Quantification of baseline Fluo-4 Ca²⁺ dye intensities for control(n=122, 4 patient lines) and HCM iPSC-CMs (n=105, 4 patient lines). (K)Representative Ca²⁺ transients of control and HCM iPSC-CMs using theIndo-1 ratiometric Ca²⁺ dye. (L) Quantification of resting Ca²⁺ levelsby measurement of Indo-1 ratio in control (n=17, 4 patient lines) andHCM iPSC-CMs (n=26, 4 patient lines). (M) Representative Ca²⁺ transienttraces from control and HCM iPSC-CMs followed by caffeine exposure. (N)Mean peak amplitudes of ΔF/F0 ratios after caffeine administrationrepresenting release of SR Ca²⁺load for control (n=23, 3 lines) and HCMiPSC-CMs (n=35, 3 lines). * denotes P<0.05 HCM vs control, ** denotesP<0.01 HCM vs control.

FIG. 33. Exacerbation of the HCM phenotype by positive inotropic stress.(A) Inotropic stimulation of control (n=50, 5 patient lines) and HCMiPSC-CMs (n=50, 5 patient lines) by the β-agonist isoproterenolaccelerated presentation of cellular hypertrophy in HCM iPSC-CMs ascompared to control counterparts. Co-administration of the β-blockerpropranolol prevented 20 catecholamine-induced hypertrophy in HCMiPSC-CMs (B) Representative Ca²⁺ line scans and waveforms in control andHCM iPSC-CMs following positive inotropic stimulation by isoproterenol.Black arrowheads indicate abnormal Ca²⁺ waveforms. (C) Quantification ofcontrol (n=50, 5 patient lines) and HCM iPSC-CMs (n=50, 5 patient lines)exhibiting irregular Ca²⁺ transients in response to treatment byisoproterenol and co-administration of propranolol. (D)Electrophysiological measurement of spontaneous action potentials andarrhythmia in control and HCM iPSC-CMs at baseline, followed by positiveinotropic stimulation by isoproterenol. Red arrows indicate DAD-likewaveforms. (E) Quantification of DAD rate in control and HCM iPSC-CMsfollowing isoproterenol administration (total DADs/total beats). *denotes P<0.05 HCM vs control, ** denotes P<0.001 HCM vs control, ##denotes P<0.01 iso+pro vs iso.

FIG. 34. Treatment of HCM iPSC-CMs by verapamil significantly mitigatesdevelopment of the HCM phenotype. (A) Representative immunostainingimages of HCM iPSC-CMs treated with 0 nM, 50 nM, and 100 nM of theL-type Ca²⁺ channel blocker verapamil for 5 continuous days beginning 25days after induction of cardiac differentiation. Quantification ofrelative cell sizes for HCM iPSC-CMs treated with verapamil (n=50, 5patient lines per treatment group). (B) Representative Ca²⁺ line scanimages and waveforms of HCM iPSC-CMs treated with 0 nM, 50 nM, and 100nM of verapamil for 5 continuous days. Quantification of percentages ofHCM iPSC-CMs found to exhibit irregular Ca²⁺ transients followingtreatment with verapamil (n=40, 5 patient lines per treatment group).(C) Representative electrophysiological recordings of spontaneous actionpotentials in HCM iPSC-CMs treated with 0 nM, 50 nM, and 100 nM ofverapamil for 5 continuous days. Quantification of DAD frequencies inHCM iPSC-CMs 21 following treatment with verapamil (n=25, 5 patientlines per treatment group). (D) Schematic for development of the HCMphenotype as caused by HCM mutations in MYH7. Red boxes indicatepotential methods to mitigate development of the disease. * denotesP<0.01 untreated vs 50 nM verapamil vs 100 nM verapamil.

The invention is best understood from the following detailed descriptionwhen read in conjunction with the accompanying drawings.

DETAILED DESCRIPTION OF THE INVENTION

Before the present compositions and methods are described, it is to beunderstood that this invention is not limited to particular compositionsand methods described, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present invention will be limited onlyby the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, some potential andpreferred methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. It is understood that the present disclosuresupersedes any disclosure of an incorporated publication to the extentthere is a contradiction.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “areprogramming factor polypeptide” includes a plurality of suchpolypeptides, and reference to “the induced pluripotent stem cells”includes reference to one or more induced pluripotent stem cells andequivalents thereof known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DEFINITIONS

Dilated cardiomyopathy (DCM) is one of the cardiomyopathies, a group ofdiseases that primarily affect the myocardium. In DCM a portion of themyocardium is dilated, often without any obvious cause. Left or rightventricular systolic pump function of the heart is impaired, leading toprogressive cardiac enlargement and hypertrophy, a process calledremodeling. Although in many cases no etiology is apparent, dilatedcardiomyopathy can result from a variety of toxic, metabolic, orinfectious agents. About 25-35% of patients have familial forms of thedisease, with most mutations affecting genes encoding cytoskeletalproteins, while some affect other proteins involved in contraction. Thedisease is genetically heterogeneous, but the most common form of itstransmission is an autosomal dominant pattern. Cytoskeletal proteinsinvolved in DCM include cardiac troponin T (TNNT2), α-cardiac actin,desmin, and the nuclear lamins A and C, and various other contractileproteins.

Hypertrophic cardiomyopathy (HCM), is a condition in which sarcomeresreplicate causing heart muscle cells to increase in size, which resultsin the thickening of the heart muscle. In addition, the normal alignmentof muscle cells is disrupted, a phenomenon known as myocardial disarray.HCM also causes disruptions of the electrical functions of the heart.HCM is most commonly due to a mutation in one of 9 sarcomeric genes thatresults in a mutated protein in the sarcomere. Myosin heavy chainmutations are associated with development of familial hypertrophiccardiomyopathy. Hypertrophic cardiomyopathy is usually inherited as anautosomal dominant trait, which mutations reported in cardiac troponin T(TNNT2); myosin heavy chain (MYH7); tropomyosin 1 (TPM1); myosin bindingprotein C (MYBPC3); 5′-AMP-activated protein kinase subunit gamma-2(PRKAG2); troponin I type 3 (TNNI3); titin (TTN); myosin, light chain 2(MYL2); actin, alpha cardiac muscle 1 (ACTC1); and cardiac LIM protein(CSRP3). An insertion/deletion polymorphism in the gene encoding forangiotensin converting enzyme (ACE) alters the clinical phenotype of thedisease. The D/D (deletion/deletion) genotype of ACE is associated withmore marked hypertrophy of the left ventricle and may be associated withhigher risk of adverse outcomes.

Anthracycline-induced cardiotoxicity (and resistance toanthracycline-induced toxicity). Anthracyclines such as doxorubicin arefrontline chemotherapeutic agents that are used to treat leukemias,Hodgkin's lymphoma, and solid tumors of the breast, bladder, stomach,lung, ovaries, thyroid, and muscle, among other organs. The primary sideeffect of anthracyclines is cardiotoxicity, which results in severeheart failure for many of the recipients receiving regimens utilizingthis chemotherapeutic agent.

Arrhythmogenic right ventricular dysplasia (ARVD). ARVD is an autosomaldominant disease of cardiac desmosomes that results in arrhythmia of theright ventricle and sudden cardiac death. It is second only tohypertrophic cardiomyopathy as a leading cause for sudden cardiac deathin the young.

Left Ventricular Non-Compaction (LVNC, aka non-compactioncardiomyopathy). LVNC is a hereditary cardiac disease which results fromimpaired development of the myocardium (heart muscle) duringembryogenesis. Patients with mutations causing LVNC develop heartfailure and abnormal cardiac electrophysiology early in life.

Double Inlet Left Ventricle (DILV). DILV is a congenital heart defect inwhich both the left and right atria feed into the left ventricle. As aresult, children born with this defect only have one functionalventricular chamber, and trouble pumping oxygenated blood into thegeneral circulation.

Long QT (Type-1) Syndrome (LQT-1, KCNQ1 mutation). Long QT syndrome(LQT) is a hereditary arrhythmic disease in which the QT phase of theelectrocardiogram is prolonged, resulting in increased susceptibilityfor arrhythmia and sudden cardiac death. There are 13 known genesassociated with LQT.

By “pluripotency” and pluripotent stem cells it is meant that such cellshave the ability to differentiate into all types of cells in anorganism. The term “induced pluripotent stem cell” encompassespluripotent cells, that, like embryonic stem (ES) cells, can be culturedover a long period of time while maintaining the ability todifferentiate into all types of cells in an organism, but that, unlikeES cells (which are derived from the inner cell mass of blastocysts),are derived from differentiated somatic cells, that is, cells that had anarrower, more defined potential and that in the absence of experimentalmanipulation could not give rise to all types of cells in the organism.iPS cells have an hESC-like morphology, growing as flat colonies withlarge nucleo-cytoplasmic ratios, defined borders and prominent nuclei.In addition, iPS cells express one or more key pluripotency markersknown by one of ordinary skill in the art, including but not limited toalkaline phosphatase, SSEA3, SSEA4, Sox2, Oct3/4, Nanog, TRA160, TRA181,TDGF 1, Dnmt3b, FoxD3, GDF3, Cyp26a1, TERT, and zfp42. In addition, theiPS cells are capable of forming teratomas. In addition, they arecapable of forming or contributing to ectoderm, mesoderm, or endodermtissues in a living organism.

As used herein, “reprogramming factors” refers to one or more, i.e. acocktail, of biologically active factors that act on a cell to altertranscription, thereby reprogramming a cell to multipotency or topluripotency. Reprogramming factors may be provided to the cells, e.g.cells from an individual with a family history or genetic make-up ofinterest for heart disease such as fibroblasts, adipocytes, etc.;individually or as a single composition, that is, as a premixedcomposition, of reprogramming factors. The factors may be provided atthe same molar ratio or at different molar ratios. The factors may beprovided once or multiple times in the course of culturing the cells ofthe subject invention. In some embodiments the reprogramming factor is atranscription factor, including without limitation, Oct3/4; Sox2; Klf4;c-Myc; Nanog; and Lin-28.

Somatic cells are contacted with reprogramming factors, as definedabove, in a combination and quantity sufficient to reprogram the cell topluripotency. Reprogramming factors may be provided to the somatic cellsindividually or as a single composition, that is, as a premixedcomposition, of reprogramming factors. In some embodiments thereprogramming factors are provided as a plurality of coding sequences ona vector.

Genes may be introduced into the somatic cells or the iPS cells derivedtherefrom for a variety of purposes, e.g. to replace genes having a lossof function mutation, provide marker genes, etc. Alternatively, vectorsare introduced that express antisense mRNA or ribozymes, therebyblocking expression of an undesired gene. Other methods of gene therapyare the introduction of drug resistance genes to enable normalprogenitor cells to have an advantage and be subject to selectivepressure, for example the multiple drug resistance gene (MDR), oranti-apoptosis genes, such as bcl-2. Various techniques known in the artmay be used to introduce nucleic acids into the target cells, e.g.electroporation, calcium precipitated DNA, fusion, transfection,lipofection, infection and the like, as discussed above. The particularmanner in which the DNA is introduced is not critical to the practice ofthe invention.

The iPS cells may also be differentiated into cardiac muscle cells.Inhibition of bone morphogenetic protein (BMP) signaling may result inthe generation of cardiac muscle cells (or cardiomyocytes), see, e.g.,Yuasa et al., (2005), Nat. Biotechnol., 23(5):607-11. Thus, in anexemplary embodiment, the induced cells are cultured in the presence ofnoggin for from about two to about six days, e.g., about 2, about 3,about 4, about 5, or about 6 days, prior to allowing formation of anembryoid body, and culturing the embryoid body for from about 1 week toabout 4 weeks, e.g., about 1, about 2, about 3, or about 4 weeks.

Cardiomyocyte differentiation may be promoted by including cardiotropicagents in the culture, such as activin A and/or bone morphogeneticprotein-4 (see the Examples herein, Xu et al. Regen Med. 2011 January;6(1):53-66; Mignone et al. Circ J. 2010 74(12):2517-26; Takei et al. AmJ Physiol Heart Circ Physiol. 2009 296(6):H1793-803, each hereinspecifically incorporated by reference). Examples of such protocols alsoinclude, for example, addition of a Wnt agonist, such as Wnt 3A,optionally in the presence of cytokines such as BMP4, VEGF and ActivinA; followed by culture in the presence of a Wnt antagonist, such asoluble frizzled protein. However, any suitable method of inducingcardiomyocyte differentiation may be used, for example, Cyclosporin Adescribed by Fujiwara et al. PLoS One. 2011 6(2):e16734; Dambrot et al.Biochem J. 2011 434(1):25-35; equiaxial cyclic stretch, angiotensin II,and phenylephrine (PE) described by Foldes et al. J Mol Cell Cardiol.2011 50(2):367-76; ascorbic acid, dimethylsulfoxide and5-aza-2′-deoxycytidine described by Wang et al. Sci China Life Sci. 201053(5):581-9, endothelial cells described by Chen et al. J Cell Biochem.2010 111(1):29-39, and the like, which are herein specificallyincorporated by reference.

The cells are harvested at an appropriate stage of development, whichmay be determined based on the expression of markers and phenotypiccharacteristics of the desired cell type e.g. at from about 1 to 4weeks. Cultures may be empirically tested by staining for the presenceof the markers of interest, by morphological determination, etc. Thecells are optionally enriched before or after the positive selectionstep by drug selection, panning, density gradient centrifugation, etc.In another embodiment, a negative selection is performed, where theselection is based on expression of one or more of markers found on EScells, fibroblasts, epithelial cells, and the like. Selection mayutilize panning methods, magnetic particle selection, particle sorterselection, and the like.

Cardiomyocytes.

Phenotypes of cardiomyocytes that arise during development of themammalian heart can be distinguished: primary cardiomyocytes; nodalcardiomyocytes; conducting cardiomyocytes and working cardiomyocytes.All cardiomyocytes have sarcomeres and a sarcoplasmic reticulum (SR),are coupled by gap junctions, and display automaticity. Cells of theprimary heart tube are characterized by high automaticity, lowconduction velocity, low contractility, and low SR activity. Thisphenotype largely persists in nodal cells. In contrast, atrial andventricular working myocardial cells display virtually no automaticity,are well coupled intercellularly, have well developed sarcomeres, andhave a high SR activity. Conducting cells from the atrioventricularbundle, bundle branches and peripheral ventricular conduction systemhave poorly developed sarcomeres, low SR activity, but are well coupledand display high automaticity.

For α-Mhc, β-Mhc and cardiac Troponin I and slow skeletal Troponin I,developmental transitions have been observed in differentiated ES cellcultures. Expression of Mlc2v and Anf is often used to demarcateventricular-like and atrial-like cells in ES cell cultures,respectively, although in ESDCs, Anf expression does not exclusivelyidentify atrial cardiomyocytes and may be a general marker of theworking myocardial cells.

A “cardiomyocyte precursor” is defined as a cell that is capable ofgiving rise to progeny that include cardiomyocytes.

In addition to various uses as an in vitro cultured cells, thecardiomyocytes may be tested in a suitable animal model. At one level,cells are assessed for their ability to survive and maintain theirphenotype in vivo. Cell compositions are administered to immunodeficientanimals (such as nude mice, or animals rendered immunodeficientchemically or by irradiation). Tissues are harvested after a period ofregrowth, and assessed as to whether the administered cells or progenythereof are still present, and may be phenotyped for response to atreatment of interest. Suitability can also be determined in an animalmodel by assessing the degree of cardiac recuperation that ensues fromtreatment with the differentiating cells of the invention. A number ofanimal models are available for such testing. For example, hearts can becryoinjured by placing a precooled aluminum rod in contact with thesurface of the anterior left ventricle wall (Murry et al., J. Clin.Invest. 98:2209, 1996; Reinecke et al., Circulation 100:193, 1999; U.S.Pat. No. 6,099,832). In larger animals, cryoinjury can be inflicted byplacing a 30-50 mm copper disk probe cooled in liquid N₂ on the anteriorwall of the left ventricle for approximately 20 min (Chiu et al., Ann.Thorac. Surg. 60:12, 1995). Infarction can be induced by ligating theleft main coronary artery (Li et al., J. Clin. Invest. 100:1991, 1997).Injured sites are treated with cell preparations of this invention, andthe heart tissue is examined by histology for the presence of the cellsin the damaged area. Cardiac function can be monitored by determiningsuch parameters as left ventricular end-diastolic pressure, developedpressure, rate of pressure rise, rate of pressure decay, etc.

The terms “treatment”, “treating”, “treat” and the like are used hereinto generally refer to obtaining a desired pharmacologic and/orphysiologic effect. The effect may be prophylactic in terms ofcompletely or partially preventing a disease or symptom thereof and/ormay be therapeutic in terms of a partial or complete stabilization orcure for a disease and/or adverse effect attributable to the disease.“Treatment” as used herein covers any treatment of a disease in amammal, particularly a human, and includes: (a) preventing the diseaseor symptom from occurring in a subject which may be predisposed to thedisease or symptom but has not yet been diagnosed as having it; (b)inhibiting the disease symptom, i.e., arresting its development; or (c)relieving the disease symptom, i.e., causing regression of the diseaseor symptom.

The terms “individual,” “subject,” “host,” and “patient,” are usedinterchangeably herein and refer to any mammalian subject for whomdiagnosis, treatment, or therapy is desired, particularly humans.

METHODS OF THE INVENTION

Methods are provided for the obtention and use of in vitro cell culturesof disease-relevant cardiomyocytes, where the cardiomyocytes aredifferentiated from induced human pluripotent stem cells (iPS cells)comprising at least one allele encoding a mutation associated with acardiac disease, as described above. Specific mutations of interestinclude, without limitation, MYH7 R663H mutation, TNNT2 R173W; PKP22013delC mutation; PKP2 Q617X mutation; and KCNQ1 G269S missensemutation. In some embodiments a panel of such cardiomyocytes areprovided, where the panel includes two or more differentdisease-relevant cardiomyocytes. In some embodiments a panel of suchcardiomyocytes are provided, where the cardiomyocytes are subjected to aplurality of candidate agents, or a plurality of doses of a candidateagent. Candidate agents include small molecules, i.e. drugs, geneticconstructs that increase or decrease expression of an RNA of interest,electrical changes, and the like.

Methods are provided for determining the activity of a candidate agenton a disease-relevant cardiomyocyte, the method comprising contactingthe candidate agent with one or a panel of cardiomyocytes differentiatedfrom induced human pluripotent stem cells (iPS cells) comprising atleast one allele encoding a mutation associated with a cardiac disease;and determining the effect of the agent on morphologic, genetic orfunctional parameters, including without limitation calcium transientamplitude, intracellular Ca²⁺ level, cell size contractile forceproduction, beating rates, sarcomeric α-actinin distribution, and geneexpression profiling.

Where the disease is DCM, the cardiomyocytes may be stimulated withpositive inotropic stress, such as a β-adrenergic agonist before, duringor after contacting with the candidate agent. In some embodiments theβ-adrenergic agonist is norepinephrine. It is shown herein that DMCcardiomyocytes have an initially positive chronotropic effect inresponse to positive inotropic stress, that later becomes negative withcharacteristics of failure such as reduced beating rates, compromisedcontraction, and significantly more cells with abnormal sarcomericα-actinin distribution. β-adrenergic blocker treatment andover-expression of sarcoplasmic reticulum Ca²⁺ ATPase (Serca2a) improvethe function. DCM cardiomyocytes may also be tested with genetic agentsin the pathways including including factors promoting cardiogenesis,integrin and cytoskeletal signaling, and ubiquitination pathway, forexample as shown in Table 8. Compared to the control healthy individualsin the same family cohort, DCM cardiomyocytes exhibit decreased calciumtransient amplitude, decreased contractility, and abnormal sarcomericα-actinin distribution.

Where the disease is HCM the cardiomyocytes may be stimulated withpositive inotropic stress, such as a β-adrenergic agonist before, duringor after contacting with the candidate agent. Under such conditions, HCMcardiomyocytes display higher hypertrophic responses, which can bereversed by a β-adrenergic blocker. Compared to healthy individuals, HCMcardiomyocytes exhibit increased cell size and up-regulation of HCMrelated genes, and more irregularity in contractions characterized byimmature beats, including a higher frequency of abnormal Ca²⁺transients, characterized by secondary immature transients. Thesecardiomyocytes have increased intracullar Ca²⁺ levels, and in someembodiments candidate agents that target calcineurin or other targetsassociated with calcium affinity.

In screening assays for the small molecules, the effect of adding acandidate agent to cells in culture is tested with a panel of cells andcellular environments, where the cellular environment includes one ormore of: electrical stimulation including alterations in ionicity, drugstimulation, and the like, and where panels of cells may vary ingenotype, in prior exposure to an environment of interest, in the doseof agent that is provided, etc., where usually at least one control isincluded, for example a negative control and a positive control. Cultureof cells is typically performed in a sterile environment, for example,at 37° C. in an incubator containing a humidified 92-95% air/5-8% CO₂atmosphere. Cell culture may be carried out in nutrient mixturescontaining undefined biological fluids such as fetal calf serum, ormedia which is fully defined and serum free. The effect of the alteringof the environment is assessed by monitoring multiple output parameters,including morphogical, functional and genetic changes.

In the screening assays for genetic agents, polynucleotides are added toone or more of the cells in a panel in order to alter the geneticcomposition of the cell. The output parameters are monitored todetermine whether there is a change in phenotype. In this way, geneticsequences are identified that encode or affect expression of proteins inpathways of interest. The results can be entered into a data processorto provide a screening results dataset. Algorithms are used for thecomparison and analysis of screening results obtained under differentconditions.

Methods for analysis include calcium imaging, where cells are loadedwith an appropriate dye and exposed to calcium in a condition ofinterest, and imaged, for example with a confocal microscope. Ca²⁺responses may be quantified, and the time-dependent Ca²⁺ response wasthen analyzed for irregularities in timing of successive Ca²⁺ transientsand for the total Ca²⁺ influx per transient. The total Ca²⁺ releasedduring each transient was determined by integrating the area underneatheach wave with respect to the baseline.

Atomic force microscopy (AFM) can be used to measure contractile forces.Beating cells are interrogated by AFM using a cantilever. To measureforces, cells are gently contacted by the cantilever tip, then thecantilever tip remains in the position for intervals while deflectiondata are collected. Statistics can be calculated for the forces,intervals between beats, and duration of each contraction for each cell.

Cells can also analyzed by microelectrode array (MEA), where beatingcardiomyocytes are plated on MEA probes, and the field potentialduration (FPD) measured and determined to provide electrophysiologicalparameters.

Methods of analysis at the single cell level are of particular interest,e.g. as described above: atomic force microscopy, microelectrode arrayrecordings, patch clamping, single cell PCR, calcium imaging, flowcytometry and the like.

Parameters are quantifiable components of cells, particularly componentsthat can be accurately measured, desirably in a high throughput system.A parameter can also be any cell component or cell product includingcell surface determinant, receptor, protein or conformational orposttranslational modification thereof, lipid, carbohydrate, organic orinorganic molecule, nucleic acid, e.g. mRNA, DNA, etc. or a portionderived from such a cell component or combinations thereof. While mostparameters will provide a quantitative readout, in some instances asemi-quantitative or qualitative result will be acceptable. Readouts mayinclude a single determined value, or may include mean, median value orthe variance, etc. Variability is expected and a range of values foreach of the set of test parameters will be obtained using standardstatistical methods with a common statistical method used to providesingle values.

Parameters of interest include detection of cytoplasmic, cell surface orsecreted biomolecules, frequently biopolymers, e.g. polypeptides,polysaccharides, polynucleotides, lipids, etc. Cell surface and secretedmolecules are a preferred parameter type as these mediate cellcommunication and cell effector responses and can be more readilyassayed. In one embodiment, parameters include specific epitopes.Epitopes are frequently identified using specific monoclonal antibodiesor receptor probes. In some cases the molecular entities comprising theepitope are from two or more substances and comprise a definedstructure; examples include combinatorially determined epitopesassociated with heterodimeric integrins. A parameter may be detection ofa specifically modified protein or oligosaccharide. A parameter may bedefined by a specific monoclonal antibody or a ligand or receptorbinding determinant.

Candidate agents of interest are biologically active agents thatencompass numerous chemical classes, primarily organic molecules, whichmay include organometallic molecules, inorganic molecules, geneticsequences, etc. An important aspect of the invention is to evaluatecandidate drugs, select therapeutic antibodies and protein-basedtherapeutics, with preferred biological response functions. Candidateagents comprise functional groups necessary for structural interactionwith proteins, particularly hydrogen bonding, and typically include atleast an amine, carbonyl, hydroxyl or carboxyl group, frequently atleast two of the functional chemical groups. The candidate agents oftencomprise cyclical carbon or heterocyclic structures and/or aromatic orpolyaromatic structures substituted with one or more of the abovefunctional groups. Candidate agents are also found among biomolecules,including peptides, polynucleotides, saccharides, fatty acids, steroids,purines, pyrimidines, derivatives, structural analogs or combinationsthereof.

Included are pharmacologically active drugs, genetically activemolecules, etc. Compounds of interest include chemotherapeutic agents,anti-inflammatory agents, hormones or hormone antagonists, ion channelmodifiers, and neuroactive agents. Exemplary of pharmaceutical agentssuitable for this invention are those described in, “The PharmacologicalBasis of Therapeutics,” Goodman and Gilman, McGraw-Hill, New York, N.Y.,(1996), Ninth edition, under the sections: Drugs Acting at Synaptic andNeuroeffector Junctional Sites; Cardiovascular Drugs; Vitamins,Dermatology; and Toxicology, all incorporated herein by reference.

Test compounds include all of the classes of molecules described above,and may further comprise samples of unknown content. Of interest arecomplex mixtures of naturally occurring compounds derived from naturalsources such as plants. While many samples will comprise compounds insolution, solid samples that can be dissolved in a suitable solvent mayalso be assayed. Samples of interest include environmental samples, e.g.ground water, sea water, mining waste, etc.; biological samples, e.g.lysates prepared from crops, tissue samples, etc.; manufacturingsamples, e.g. time course during preparation of pharmaceuticals; as wellas libraries of compounds prepared for analysis; and the like. Samplesof interest include compounds being assessed for potential therapeuticvalue, i.e. drug candidates.

The term samples also includes the fluids described above to whichadditional components have been added, for example components thataffect the ionic strength, pH, total protein concentration, etc. Inaddition, the samples may be treated to achieve at least partialfractionation or concentration. Biological samples may be stored if careis taken to reduce degradation of the compound, e.g. under nitrogen,frozen, or a combination thereof. The volume of sample used issufficient to allow for measurable detection, usually from about 0.1:Ito 1 ml of a biological sample is sufficient.

Compounds, including candidate agents, are obtained from a wide varietyof sources including libraries of synthetic or natural compounds. Forexample, numerous means are available for random and directed synthesisof a wide variety of organic compounds, including biomolecules,including expression of randomized oligonucleotides and oligopeptides.Alternatively, libraries of natural compounds in the form of bacterial,fungal, plant and animal extracts are available or readily produced.Additionally, natural or synthetically produced libraries and compoundsare readily modified through conventional chemical, physical andbiochemical means, and may be used to produce combinatorial libraries.Known pharmacological agents may be subjected to directed or randomchemical modifications, such as acylation, alkylation, esterification,amidification, etc. to produce structural analogs.

As used herein, the term “genetic agent” refers to polynucleotides andanalogs thereof, which agents are tested in the screening assays of theinvention by addition of the genetic agent to a cell. The introductionof the genetic agent results in an alteration of the total geneticcomposition of the cell. Genetic agents such as DNA can result in anexperimentally introduced change in the genome of a cell, generallythrough the integration of the sequence into a chromosome. Geneticchanges can also be transient, where the exogenous sequence is notintegrated but is maintained as an episomal agents. Genetic agents, suchas antisense oligonucleotides, can also affect the expression ofproteins without changing the cell's genotype, by interfering with thetranscription or translation of mRNA. The effect of a genetic agent isto increase or decrease expression of one or more gene products in thecell.

Introduction of an expression vector encoding a polypeptide can be usedto express the encoded product in cells lacking the sequence, or toover-express the product. Various promoters can be used that areconstitutive or subject to external regulation, where in the lattersituation, one can turn on or off the transcription of a gene. Thesecoding sequences may include full-length cDNA or genomic clones,fragments derived therefrom, or chimeras that combine a naturallyoccurring sequence with functional or structural domains of other codingsequences. Alternatively, the introduced sequence may encode ananti-sense sequence; be an anti-sense oligonucleotide; RNAi, encode adominant negative mutation, or dominant or constitutively activemutations of native sequences; altered regulatory sequences, etc.

Antisense and RNAi oligonucleotides can be chemically synthesized bymethods known in the art. Preferred oligonucleotides are chemicallymodified from the native phosphodiester structure, in order to increasetheir intracellular stability and binding affinity. A number of suchmodifications have been described in the literature, which alter thechemistry of the backbone, sugars or heterocyclic bases. Among usefulchanges in the backbone chemistry are phosphorothioates;phosphorodithioates, where both of the non-bridging oxygens aresubstituted with sulfur; phosphoroamidites; alkyl phosphotriesters andboranophosphates. Achiral phosphate derivatives include3′-O′-5′-S-phosphorothioate, 3′-S-5′-O-phosphorothioate,3′-CH2-5′-O-phosphonate and 3′-NH-5′-O-phosphoroamidate. Peptide nucleicacids replace the entire ribose phosphodiester backbone with a peptidelinkage. Sugar modifications are also used to enhance stability andaffinity, e.g. morpholino oligonucleotide analogs. The □-anomer ofdeoxyribose may be used, where the base is inverted with respect to thenatural □-anomer. The 2′-OH of the ribose sugar may be altered to form2′-O-methyl or 2′-O-allyl sugars, which provides resistance todegradation without comprising affinity.

Agents are screened for biological activity by adding the agent to atleast one and usually a plurality of cells, in one or in a plurality ofenvironmental conditions, e.g. following stimulation with a β-adrenergicagonist, following electric or mechanical stimulation, etc. The changein parameter readout in response to the agent is measured, desirablynormalized, and the resulting screening results may then be evaluated bycomparison to reference screening results, e.g. with cells having othermutations of interest, normal cardiomyocytes, cardiomyocytes derivedfrom other family members, and the like. The reference screening resultsmay include readouts in the presence and absence of differentenvironmental changes, screening results obtained with other agents,which may or may not include known drugs, etc.

The agents are conveniently added in solution, or readily soluble form,to the medium of cells in culture. The agents may be added in aflow-through system, as a stream, intermittent or continuous, oralternatively, adding a bolus of the compound, singly or incrementally,to an otherwise static solution. In a flow-through system, two fluidsare used, where one is a physiologically neutral solution, and the otheris the same solution with the test compound added. The first fluid ispassed over the cells, followed by the second. In a single solutionmethod, a bolus of the test compound is added to the volume of mediumsurrounding the cells. The overall concentrations of the components ofthe culture medium should not change significantly with the addition ofthe bolus, or between the two solutions in a flow through method.

Preferred agent formulations do not include additional components, suchas preservatives, that may have a significant effect on the overallformulation. Thus preferred formulations consist essentially of abiologically active compound and a physiologically acceptable carrier,e.g. water, ethanol, DMSO, etc. However, if a compound is liquid withouta solvent, the formulation may consist essentially of the compounditself.

A plurality of assays may be run in parallel with different agentconcentrations to obtain a differential response to the variousconcentrations. As known in the art, determining the effectiveconcentration of an agent typically uses a range of concentrationsresulting from 1:10, or other log scale, dilutions. The concentrationsmay be further refined with a second series of dilutions, if necessary.Typically, one of these concentrations serves as a negative control,i.e. at zero concentration or below the level of detection of the agentor at or below the concentration of agent that does not give adetectable change in the phenotype.

Various methods can be utilized for quantifying the presence of selectedparameters, in addition to the functional parameters described above.For measuring the amount of a molecule that is present, a convenientmethod is to label a molecule with a detectable moiety, which may befluorescent, luminescent, radioactive, enzymatically active, etc.,particularly a molecule specific for binding to the parameter with highaffinity Fluorescent moieties are readily available for labelingvirtually any biomolecule, structure, or cell type. Immunofluorescentmoieties can be directed to bind not only to specific proteins but alsospecific conformations, cleavage products, or site modifications likephosphorylation. Individual peptides and proteins can be engineered toautofluoresce, e.g. by expressing them as green fluorescent proteinchimeras inside cells (for a review see Jones et al. (1999) TrendsBiotechnol. 17(12):477-81). Thus, antibodies can be genetically modifiedto provide a fluorescent dye as part of their structure

Depending upon the label chosen, parameters may be measured using otherthan fluorescent labels, using such immunoassay techniques asradioimmunoassay (RIA) or enzyme linked immunosorbance assay (ELISA),homogeneous enzyme immunoassays, and related non-enzymatic techniques.These techniques utilize specific antibodies as reporter molecules,which are particularly useful due to their high degree of specificityfor attaching to a single molecular target. U.S. Pat. No. 4,568,649describes ligand detection systems, which employ scintillation counting.These techniques are particularly useful for protein or modified proteinparameters or epitopes, or carbohydrate determinants. Cell readouts forproteins and other cell determinants can be obtained using fluorescentor otherwise tagged reporter molecules. Cell based ELISA or relatednon-enzymatic or fluorescence-based methods enable measurement of cellsurface parameters and secreted parameters. Capture ELISA and relatednon-enzymatic methods usually employ two specific antibodies or reportermolecules and are useful for measuring parameters in solution. Flowcytometry methods are useful for measuring cell surface andintracellular parameters, as well as shape change and granularity andfor analyses of beads used as antibody- or probe-linked reagents.Readouts from such assays may be the mean fluorescence associated withindividual fluorescent antibody-detected cell surface molecules orcytokines, or the average fluorescence intensity, the medianfluorescence intensity, the variance in fluorescence intensity, or somerelationship among these.

Both single cell multiparameter and multicell multiparameter multiplexassays, where input cell types are identified and parameters are read byquantitative imaging and fluorescence and confocal microscopy are usedin the art, see Confocal Microscopy Methods and Protocols (Methods inMolecular Biology Vol. 122.) Paddock, Ed., Humana Press, 1998. Thesemethods are described in U.S. Pat. No. 5,989,833 issued Nov. 23, 1999.

The quantitation of nucleic acids, especially messenger RNAs, is also ofinterest as a parameter. These can be measured by hybridizationtechniques that depend on the sequence of nucleic acid nucleotides.Techniques include polymerase chain reaction methods as well as genearray techniques. See Current Protocols in Molecular Biology, Ausubel etal., eds, John Wiley & Sons, New York, N.Y., 2000; Freeman et al. (1999)Biotechniques 26(1):112-225; Kawamoto et al. (1999) Genome Res9(12):1305-12; and Chen et al. (1998) Genomics 51(3):313-24, forexamples.

The comparison of a screening results obtained from a test compound, anda reference screening results(s) is accomplished by the use of suitablededuction protocols, AI systems, statistical comparisons, etc.Preferably, the screening results is compared with a database ofreference screening results. A database of reference screening resultscan be compiled. These databases may include reference results frompanels that include known agents or combinations of agents, as well asreferences from the analysis of cells treated under environmentalconditions in which single or multiple environmental conditions orparameters are removed or specifically altered. Reference results mayalso be generated from panels containing cells with genetic constructsthat selectively target or modulate specific cellular pathways.

The readout may be a mean, average, median or the variance or otherstatistically or mathematically derived value associated with themeasurement. The parameter readout information may be further refined bydirect comparison with the corresponding reference readout. The absolutevalues obtained for each parameter under identical conditions willdisplay a variability that is inherent in live biological systems andalso reflects individual cellular variability as well as the variabilityinherent between individuals.

For convenience, the systems of the subject invention may be provided inkits. The kits could include the cells to be used, which may be frozen,refrigerated or treated in some other manner to maintain viability,reagents for measuring the parameters, and software for preparing thescreening results. The software will receive the results and performanalysis and can include reference data. The software can also normalizethe results with the results from a control culture. The composition mayoptionally be packaged in a suitable container with written instructionsfor a desired purpose, such as screening methods, and the like.

For further elaboration of general techniques useful in the practice ofthis invention, the practitioner can refer to standard textbooks andreviews in cell biology, tissue culture, embryology, andcardiophysiology. With respect to tissue culture and embryonic stemcells, the reader may wish to refer to Teratocarcinomas and embryonicstem cells: A practical approach (E. J. Robertson, ed., IRL Press Ltd.1987); Guide to Techniques in Mouse Development (P. M. Wasserman et al.eds., Academic Press 1993); Embryonic Stem Cell Differentiation in Vitro(M. V. Wiles, Meth. Enzymol. 225:900, 1993); Properties and uses ofEmbryonic Stem Cells: Prospects for Application to Human Biology andGene Therapy (P. D. Rathjen et al., Reprod. Fertil. Dev. 10:31, 1998).With respect to the culture of heart cells, standard references includeThe Heart Cell in Culture (A. Pinson ed., CRC Press 1987), IsolatedAdult Cardiomyocytes (Vols. I & II, Piper & Isenberg eds, CRC Press1989), Heart Development (Harvey & Rosenthal, Academic Press 1998).

General methods in molecular and cellular biochemistry can be found insuch standard textbooks as Molecular Cloning: A Laboratory Manual, 3rdEd. (Sambrook et al., Harbor Laboratory Press 2001); Short Protocols inMolecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); NonviralVectors for Gene Therapy (Wagner et al. eds., Academic Press 1999);Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); ImmunologyMethods Manual (I. Lefkovits ed., Academic Press 1997); and Cell andTissue Culture: Laboratory Procedures in Biotechnology (Doyle &Griffiths, John Wiley & Sons 1998). Reagents, cloning vectors, and kitsfor genetic manipulation referred to in this disclosure are availablefrom commercial vendors such as BioRad, Stratagene, Invitrogen,Sigma-Aldrich, and ClonTech.

Each publication cited in this specification is hereby incorporated byreference in its entirety for all purposes.

It is to be understood that this invention is not limited to theparticular methodology, protocols, cell lines, animal species or genera,and reagents described, as such may vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention, which will be limited only by the appendedclaims.

As used herein the singular forms “a”, “and”, and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a cell” includes a plurality of such cells andreference to “the culture” includes reference to one or more culturesand equivalents thereof known to those skilled in the art, and so forth.All technical and scientific terms used herein have the same meaning ascommonly understood to one of ordinary skill in the art to which thisinvention belongs unless clearly indicated otherwise.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

EXAMPLES Example 1

Dilated cardiomyopathy (DCM) is the most common cardiomyopathy,characterized by ventricular dilatation, systolic dysfunction, andprogressive heart failure. DCM is the most common diagnosis leading toheart transplantation and places a considerable burden on healthcareworldwide. Here we generated cardiomyocytes (CMs) from iPSCs derivedfrom patients of a DCM family carrying a point mutation (R173W) in thegene that encodes sarcomeric protein cardiac troponin T. Compared to thecontrol healthy individuals in the same family cohort, DCM iPSC-CMsexhibited decreased calcium transient amplitude, decreasedcontractility, and abnormal sarcomeric α-actinin distribution. Whenstimulated with β-adrenergic agonist, DCM iPSC-CMs showedcharacteristics of failure such as reduced beating rates, compromisedcontraction, and significantly more cells with abnormal sarcomericα-actinin distribution. β-adrenergic blocker treatment andover-expression of sarcoplasmic reticulum Ca²⁺ ATPase (Serca2a) improvedthe function of DCM iPSC-CMs. Our study demonstrated that human DCMiPSC-CMs recapitulated the disease phenotypes morphologically andfunctionally, and thus can serve as a useful platform for exploringmolecular and cellular mechanisms and optimizing treatment of thisparticular disease.

We recruited a cohort of seven individuals from a DCM proband carryingan autosomal dominant point mutation on exon 12 of the gene TNNT2, whichcauses an Arginine (R) to Tryptophan (W) mutation at amino acid position173 in the protein cTnT. The causal effect for DCM of this particularpoint mutation was confirmed by genetic screening of a panel of 17primary DCM associated genes (Table 1) and genetic co-segregationstudies (Table 2). This mutation was also reported in a completelyindependent Belgian family. The seven recruited individuals covered 3generations (I, II, and III) (FIG. 1a ). Four patients (Ia, IIa, IIb,and IIIa) were confirmed to carry the TNNT2 R173W mutation in one of thetwo alleles by PCR amplifying the genomic locus of TNNT2 and DNAsequencing, while the other 3 individuals (Ib, IIc, and IIIb) wereconfirmed normal and served as controls in the subsequent studies (FIG.1b ). All four patients who carry the specific R173W mutation manifestedclinical DCM symptoms with dilated left ventricle and decreased ejectionfraction, and were treated medically (Table 2). A 14-year-old diseasedpatient (IIIa) had an orthotopic heart transplant due to severe clinicalsymptoms. Further genetic screening by exome sequencing of thisparticular patient IIIa with a panel of 32 most updated DCM-associatedgenes did not find any other additional variants that associate with thedisease (Table 3).

To generate patient-specific iPSCs for the seven individuals, skinfibroblasts were expanded from skin biopsies taken from each individual(FIG. 1c ) and reprogrammed with lentiviral Yamanaka 4 factors (Oct4,Sox2, Klf4, and c-MYC) under feeder-free condition. Colonies withTRA-1-60⁺ staining and human embryonic stem cell (hESC)-like morphology(FIGS. 1d and 1e ) were picked, expanded, and established as individualiPSC lines. For each individual, 3-4 iPSC lines were established forsubsequent analyses. All of the DCM iPSC lines were confirmed to containthe specific R173W mutation by genomic PCR and DNA sequencing (FIG. 5).All established iPSC lines expressed the pluripotency markers Oct4,Nanog, TRA-1-81, and SSEA-4, and were positive for alkaline phosphatase(FIG. 1f ). Microarray analyses indicated these iPSC lines were distinctfrom the parental skin fibroblasts, expressing a global gene patternmore similar to hESCs (FIG. 6). Quantitative bisulphite sequencingshowed that the promoter regions of Oct4 and Nanog were hypomethylatedin all the tested iPSC lines, indicating active transcription of thepluripotency genes (FIG. 1g ). The established iPSC lines maintained anormal karyotype after extended passage (FIG. 7) and the majority ofthem exhibited silencing of exogenous transgenes and re-expression ofendogenous Nanog (FIG. 8). iPSC lines with incomplete transgenesilencing were removed from the subsequent studies. Thesepatient-specific iPSCs were able to differentiate in vitro into cells ofall three germ layers (FIG. 9) and form teratomas following injectioninto the kidney capsules of immunodeficient mice (FIG. 1h ).

We next differentiated the DCM iPSCs into the cardiovascular lineageusing a well-established 3D differentiation protocol developed by Yang,L., et al. Human cardiovascular progenitor cells develop from a KDR+embryonic-stem-cell-derived population. Nature 453, 524-528 (2008). TwoiPSC lines from each individual were selected to be differentiated intospontaneous beating embryoid bodies (EBs). Spontaneous beating wasobserved as early as day 8 post differentiation. The efficiency ofdifferentiation to cardiac lineage varied among different lines (FIG.10). Beating EBs derived from control and patient iPSCs containedapproximately 50-60% cTnT positive CMs (FIG. 11). Allele-specific PCR ofbeating EBs derived from three iPSC clones of 3 DCM patients indicatedbi-allelic expression of the wild type and mutant (R173W) TNNT2 gene(FIG. 12). The beating EBs from the control iPSCs and DCM iPSCs wereseeded on multi-electrode array (MEA) probe (FIG. 13a ) and theirelectrophysiological properties recorded (FIG. 13b ). Both control(n=45) and DCM (n=57) iPSC-derived beating EBs exhibited comparable beatfrequencies, field potentials, interspike intervals, and field potentialdurations (FPD) at baseline (Table 4 and FIG. 13c ).

We next dissociated the beating EBs into small beating clusters andsingle beating CMs for further analyses. Single cell PCR analysis usingmicrofluidics technology on control (n=24) and DCM (n=24) iPSC-CMsindicated that there are no significant differences in the geneexpression of the selected cardiac-related transcription factors,sarcomeric proteins, and ion channels (FIG. 14). We next assessed theorganization of myofibrils in the iPSC-CMs by immunocytochemistry. Bothcontrol and DCM iPSC-CMs expressed sarcomeric proteins cTnT, sarcomericα-actinin, and myosin light chain 2a (MLC2a), as well as the cardiacmarker gap junction protein connexin 43 (FIG. 15). However, compared tocontrol iPSC-CMs (n=368) at day 30 post differentiation, a significanthigher percentage of DCM iPSC-CMs (n=391) showed a punctate distributionof sarcomeric α-actinin over one fourth of the total cellular area(p=0.008) (FIG. 2a, 2b and FIG. 16). There were no significantdifferences in cell size between control and DCM iPSC-CMs (FIG. 2c ) atthis stage. This phenotype was consistently observed in two differentDCM iPSC lines each from the 4 DCM patients, suggesting a homogeneouscorrelation to the disease-causing R173W mutation. Notably, the majorityof CMs with punctate sarcomeric α-actinin distribution were single cellsor cells at the periphery of a beating cluster. Sarcomeric α-actinin isan excellent marker for sarcomeric integrity and degeneration. Theseresults also suggest a higher tendency for individual DCM iPSC-CMs tomalfunction in maintaining sarcomere integrity.

Positive inotropic stress can induce DCM phenotype in transgenic mousemodels of DCM and aggravate the disease in clinical patients. We nextexamined whether treatment with positive inotropic reagent, such asβ-adrenergic agonists, can expedite the phenotypic response of DCMiPSC-CMs. Indeed, 10 μM norepinephrine (NE) treatment induced an initialpositive chronotropic effect that later became negative, eventuallyleading to the failure of spontaneous contraction in DCM iPSC-derivedbeating EBs (n=14) as reflected by MEA recording. By contrast, thecontrol iPSC-derived beating EBs (n=14) exhibited prolonged positivechronotropic activities (FIG. 2d ). One week of NE treatment markedlyincreased the number of CMs with punctate sarcomeric α-actinindistribution from DCM iPSC clones, with almost 90% of the DCM iPSC-CMsfound to have the disorganized sarcomeric pattern (FIG. 2f, 2g , FIGS.17 and 18). A few single DCM iPSC-CMs showed complete degeneration ofmyofilaments after prolonged NE treatment, which was not observed incontrol cells. Tracking with video imaging of individual beatingclusters of both control and DCM iPSC-CMs treated with NE over timeshowed distinct outcomes. Decreased inotropic and chronotropicactivities were observed in the DCM iPSC-CMs, but not in the controliPSC-CMs (FIG. 2e, 2h ). Single cell PCR analysis also revealed distinctgene expression changes in DCM (n=16) versus control iPSC-CMs (n=16)after NE treatment (FIG. 19). These results indicated that β-adrenergicstimulation aggravated the phenotype of DCM iPSC-CMs.

CM contraction starts from the electrical excitation of the myocytes, asreflected by the membrane action potentials (APs). To investigate thepossible underlying mechanism of DCM, we assessed whether the DCM-linkedR173W mutation in cTnT affects the electrical excitation of the CMs. Weexamined the electrical activities of the dissociated single beatingiPSC-CMs by patch clamping. Three types of spontaneous APs(ventricular-like, atrial-like, and nodal-like) were observed in bothcontrol and DCM iPSC-CMs (FIG. 20a ). DCM ventricular myocytes (n=17)exhibited normal APs that were comparable to control (n=18) (FIG. 20b ).The average action potential duration at 90% repolarization (APD90) ofthe DCM iPSC-CMs was not significantly different from that seen incontrol iPSC-CMs (FIG. 20c ). The average AP frequency, peak amplitude,and resting potential were also very similar between the 2 groups (FIGS.20d, 20e, and 20f ). These results indicated that the electricalexcitation activities of control and DCM iPSC-CMs at baseline werenormal.

To further investigate the underlying DCM disease mechanism, we measuredthe Ca²⁺ handling properties at the excitation-contraction couplinglevel by fluorescent Ca²⁺ imaging. DCM iPSC-CMs (n=40) exhibitedrhythmic frequency, timing, and amplitude of global [Ca²⁺], transientscomparable to those of the control iPSC-CMs (n=87) (FIGS. 3a, 3b, 3c,3e, and 3f ). However, DCM iPSC-CMs exhibited significantly smaller[Ca²⁺], transient amplitudes compared to those of the control iPSC-CMs(p=0.002) (FIG. 3d ), indicating the [Ca²⁺]_(i) available for eachcontraction of DCM iPSC-CMs was significantly lower. The smaller[Ca²⁺]_(i) transients of CMs were consistently observed in all examinedDCM iPSC lines derived from the 4 DCM patients, suggesting weaker forceproduction in DCM iPSC-CMs.

Deficiency in contractile force production is one of the most importantmechanisms responsible for inducing DCM and heart failure. To furtherinvestigate this, we next measured the contraction force of iPSC-CMsusing atomic force microscopy (AFM), which has been used to measurecultured chicken embryonic CMs. The AFM allowed us to probe thecontractile properties at a single cell level (FIG. 21). Compared tosingle control iPSC-CMs (n=13), DCM iPSC-CMs (n=17) showed similar beatfrequency and duration but significantly weaker contraction forces (FIG.4c, 4d , FIG. 22, and Table 5). There was no correlation between thecell size and contraction force from each single cell measured by AFM(FIG. 23).

Previous studies have shown that Serca2a over-expression, a treatmentinvestigated in a pre-clinical trial, mobilized intracellular Ca²⁺ andrestored contractility of cardiomyocytes in failing human hearts andimproved failing heart functions in animal models. Given our resultsshowing smaller Ca²⁺ transients and compromised contractility in DCMiPSC-CMs, we hypothesized that over-expression of Serca2a can rescue thephenotypes of DCM iPSC-CMs. Transduction of DCM iPSC-CMs withadenoviruses carrying Serca2a co-expressing GFP (Ad.Seca2a) (see Methodssection) at a multiplicity of infection (MOI) of 100 led toover-expression of Serca2a in these cells (FIG. 4a ). Compared to DCMiPSC-CMs transduced with adenoviruses carrying GFP only (Ad.GFP) (MOI100), over-expression of Serca2a resulted in a higher number ofspontaneous contraction foci in vitro over time (FIG. 24). Co-expressionof GFP along with Serca2a allowed us to recognize the transduced cellsand measure the contractile force by AFM (FIG. 4b ). Over-expression ofSerca2a (n=12) restored the contractile force of single DCM iPSC-CMs toa level similar to that seen in control iPSC-CMs (FIG. 4c, 4d , andTable 5), but without improvement in sarcomeric organization (FIG. 4g ).Calcium imaging using the red fluorescent Ca²⁺ indicator Rhod-2 AM (FIG.25) indicated that DCM iPSC-CMs transduced with Ad.Serca2a co-expressingGFP (n=22) had significantly increased global [Ca²⁺]_(i) transientscompared to cells transduced with Ad.GFP only (n=14) (FIGS. 4e and 4f )(p=0.04), which is consistent with the restored force production. Bycontrast, over-expression of Serca2a in control iPSC-CMs failed toproduce a statistically significant increase in contractility (FIG. 26),suggesting natural differences in calcium handling between control andDCM iPSC-CMs. Altogether, these results indicated that over-expressionof Serca2a increased the [Ca²⁺]_(i) transients and contraction force ofDCM iPSC-CMs and improved their function.

Although Serca2a gene therapy is now in clinical trial, the overallmechanism of individual CM cellular response after Serca2a gene therapyhas not been extensively studied previously. Hence we set out toinvestigate the mechanisms in which Serca2a delivery restores defects inDCM iPSC-CMs. Gene expression profiling of DCM iPSC-CMs after Serca2aover-expression showed that 191 genes (65 upregulated and 126downregulated) had greater than 1.5 fold expression changes and wererescued to an expression level similar to those in control iPSC-CMs(FIG. 27a ). Enriched pathways analysis indicated that severalpreviously known pathways, such as calcium signaling, protein kinase Asignaling, and G-protein coupled receptor signaling, are significantlyinvolved in rescuing the DCM phenotype by Serca2a over-expression.Interestingly, several pathways not previously linked to DCM, includingfactors promoting cardiogenesis, integrin and cytoskeletal signaling,and ubiquitination pathway, were also found to participate in rescuingthe DCM CM function (FIG. 27b and Table 7).

Clinical studies have shown that metoprolol, a β1-selective β-adrenergicblocker, has a beneficial effect on the clinical symptoms andhemodynamic status of DCM patients. When treated with 10 μM metoprolol,DCM iPSC-CMs showed an improvement in the sarcomeric organization asindicated by sarcomeric α-actinin staining (FIG. 28a ). Metoprololtreatment also prevented aggravation of the DCM iPSC-CMs that is inducedby NE treatment (FIG. 28b ). We observed no significant effect onsarcomeric α-actinin distribution in control iPSC-CMs treated withmetoprolol (FIG. 28c ). These results suggest that blockade ofβ-adrenergic pathway helped DCM iPSC-CMs resist mechanicaldeterioration.

In summary, we have generated patient-specific iPSCs from a DCM familycarrying a single point mutation in the sarcomeric protein cTnT andderived CMs from these iPSCs. This has allowed us to generate a largenumber of DCM-specific CMs and to analyze their functional properties,explore the underlying disease mechanisms, and test effective therapies(Table 8). Although the baseline electrophysiological activities of theDCM iPSC-CMs were not significantly different from those of thecontrols, DCM iPSC-CMs exhibited significantly smaller [Ca²⁺]_(i)transients and decreased contractile force. A weaker ability to resistmechanical stimulation was also associated with DCM iPSC-CMs. NEstimulation induced a cessation of their spontaneous contraction andmarkedly exacerbated sarcomeric organization as reflected by sarcomericα-actinin staining. This TNNT2 R173W mutation seems to affect only theCM function and not other cells from cardiovascular lineage (FIG. 29).Taken together, our data indicated that the TNNT2 R173W mutation causedimpairment in force production of CMs, which might be the primary reasonfor the eventual appearance of the DCM clinical phenotype in patients(FIG. 30). We showed both β-blocker metoprolol can rescue the DCMiPSC-CM phenotype. In addition, over-expression with Serca2a, a novelgene therapy treatment for heart failure currently in clinical trials,can significantly improve the function of DCM iPSC-CMs. Gene expressionprofiling further identified several novel pathways, includingubiquitination and integrin signaling pathways, that are involved inSerca2a rescue. Taken together, our findings demonstrate that the iPSCplatform opens new, exciting areas of research on investigating diseasemechanisms and therapeutic targets for DCM.

Methods

Patient-specific iPSC derivation, culture, and characterization. All theprotocols for this study were approved by the Stanford University HumanSubjects Research Institutional Review Board. Generation, maintenance,and characterization of patient-specific iPSC lines were performed aspreviously described.

Immunofluorescence and alkaline phosphatase staining. Alkalinephosphatase (AP) staining was performed using the Quantitative AlkalinePhosphatase ES Characterization Kit (Chemicon) following themanufacturer's instruction. Immunofluorescence was performed usingappropriate primary antibodies and AlexaFluor conjugated secondaryantibodies (Invitrogen) as previously described. The primary antibodiesfor Oct3/4 (Santa Cruz), Sox2 (Biolegend), SSEA-3 (Millipore), SSEA-4(Millipore), Tra-1-60 (Millipore), Tra-1-81 (Millipore), Nanog (SantaCruz), AFP (Santa Cruz), smooth muscle actin (SMA) (Sigma), Tuj-1(Covance), cTnT (Thermo Scientific), sarcomeric α-actinin (Clone EA-53,Sigma), Connexin-43 (Millipore), and Myosin light chain (MLC-2a)(Synaptic Systems) were used in this study.

Bisulphite pyrosequencing. One μg of sample DNA was bisulfate treatedusing the Zymo DNA Methylation Kit (Zymo Research) following themanufacturer's instruction. The PCR product was then converted tosingle-stranded DNA templates and sequenced by Pyrosequencing PSQ96 HSSystem (Biotage). The methylation status of each locus was analyzedindividually as a T/C SNP using QCpG software (Biotage).

Cardiac differentiation of human ESCs and iPSCs. Differentiation of H7ESCs and derived iPSC lines into the cardiac lineage was performed usingthe well established protocol described by Yang et al. Beating EBs weredissociated with type I collagenase (Sigma) and seeded on gelatin coatedculture dishes, glass chamber slides, or glass coverslips for functionalanalyses.

Calcium imaging. Dissociated iPSC-CMs were seeded in gelatin-coated4-well LAB-TEK® II chambers (Nalge Nunc International, chamber #1.5German coverglass system) for calcium imaging. Cells were loaded with 5μM Fluo-4 AM or Rhod-2 AM (for cells expressing GFP) and 0.02% PluronicF-127 (all from Molecular Probes) in the Tyrodes solution (140 mM NaCl,5.4 mM KCl, 1 mM MgCl₂, 10 mM glucose, 1.8 mM CaCl₂, and 10 mM HEPES pH7.4 with NaOH at 25° C.) for 15 min at 37° C. Cells were then washedthree times with the Tyrodes solution. Calcium imaging was conductedwith a confocal microscope (Carl Zeiss, LSM 510 Meta) with a 63× lens(NA=1.4) using Zen software. Spontaneous Ca²⁺ transients were acquiredat room temperature using line scan mode at a sampling rate of 1.92ms/line. A total of 10,000 lines were acquired for 19.2 s recoding.

Analysis of calcium imaging traces. Ca²⁺ responses were quantified usingFiji, a derivative of ImageJ (National Institutes of Health) to averagethe fluorescence intensity of each line. The time-dependent Ca²⁺response was then analyzed for irregularities in timing of successiveCa²⁺ transients and for the total Ca²⁺ influx per transient usingMATLAB. Time between transients (timing) was defined as the time betweenthe peaks of two successive spikes. The spikes were determined bycalculating the zero crossing of the second derivative using MATLAB'sSignal Processing Toolbox. The total Ca²⁺ released during each transientwas determined by integrating the area underneath each wave with respectto the baseline. The baseline was defined as the median of all minima.Irregularity for both spike timing and amplitude was defined as theratio of the standard deviation (s.d.) to the mean of a set ofmeasurements.

Atomic force microscopy (AFM). iPSC-CMs were seeded on glass bottompetri dishes before each experiment, switched from culture media to warmTyrode's solution. Cells were maintained at 36° C. for the entireexperiment. Beating cells were interrogated by AFM (MFP-3D Bio, AsylumResearch) using a silicon nitride cantilever (spring constants ˜0.04N/m, BudgetSensors). To measure forces, cells were gently contacted bythe cantilever tip with 100 pN of force, with a typical cellularindentation of around 100-200 nm, then the cantilever tip remained inthe position without Z-piezo feedback for multiple sequential two minuteintervals while deflection data were collected at a sample rate of 2kHz. Typical noise during these measurements was around 20 pN.Deflection data were converted to force by multiplying by the springconstant. Typically, 100-400 beats were collected for each single cell,and statistics were calculated for the forces, intervals between beats,and duration of each contraction for each cell. Forces across cells werecompared using two tailed Student's t-test.

Adenovirus transduction of iPSC-CMs. First-generation type 5 recombinantadenoviruses carrying cytomegalovirus (CMV) promoter driving Serca2aplus a separate CMV promoter driving GFP (Ad.Serca2a) and adenovirusescarrying CMV promoter driving GFP only (Ad.GFP) as control were used.iPSC-CMs dissociated from beating EBs were transduced at MOI 100overnight and then refreshed with culture medium (DMEM supplemented with10% FBS). Cells were used for subsequent experiments 48 hours aftertransduction.

Statistical analysis. Data were analyzed using either Excel or R.Statistical differences among two groups were tested using two tailedStudent's t-tests. Statistical differences among more than two groupswere analyzed using one-way ANOVA tests followed by Tukey's MultipleComparison Test. Significant differences were determined when p value isless than 0.05.

Genetic testing. Peripheral blood was drawn in EDTA from the patientsand sent to GeneDx Laboratories (Gaithersburg, Md.) for isolation ofgenomic DNA and commercial genetic testing. The DNA was amplified andsequenced using a “next generation” solid-state sequence-by synthesismethod (Illumine). The DCM gene panel includes sequencing of thecomplete coding regions and splice junctions of the following genes:LMNA, MYH7, TNNT2, ACTC1, DES, MYBPC3, TPM1, TNNI3, LDB3, TAZ, PLN, TTR,LAMP2, SGCD, MTTL1, MTTQ, MTTH, MTTK, MTTS1, MTTS2, MTND1, MTND5, andMTND6. Results were compared with the human reference sequence (c DNANM_00108005). Possible disease associated sequences were confirmed bydideoxy DNA sequencing. The presence of candidate disease associatedvariants was also determined in 335 presumed healthy controls of mixedethnicity. A variant was identified (p.Arg173Trp) in the TNNT2 gene.This is a non-conservative amino acid substitution of a hydrophilic,positive arginine with a hydrophobic, neutral tyrosine. This arginine ishighly conserved at position 173 across several species. In silicoanalysis (PolyPhen) predicts the amino acid substitution to be damagingto the cardiac troponin T2 protein structure and function. This variantwas not found in 335 control individuals of mixed descent.

Exome sequencing and data analysis. Genomic DNA from individual IIIa wassubjected to exome sequencing using the Nimblegen SeqCap EZ ExomeLibrary v2.0 (Roche Molecular Biochemicals). Thirty two most updatedautosomal genes underlying DCM¹ (Supplementary Table 3) were examinedfor mutations. All of these genes were targeted in the exome capture andin total covered 190 kb. Sequencing with one lane of HiSeq (Illumina)generated median coverage of 217× (interquartile range 152× to 243×).Single nucleotide variant (SNVs) were found by using an analysispipeline comprised of Novoalign, Picard, SAMtools, GATK, and ANNOVAR.SNPs were confirmed by comparing the SNPs data base dbSNP132.Deleterious SNVs were identified by the SIFT algorithm.

Microarray hybridization and data analysis. Total RNA samples frombiological duplicates of of undifferentiated iPSCs or 4-week-old CMsderived from control and DCM iPSCs (treated with or without Serca2aover-expression) were hybridized to Affymetrix GeneChip Human Gene 1.0ST Array, and then normalized and annotated by the Affymetrix ExpressionConsole software. The Pearson Correlation Coefficient was calculated foreach pair of samples using the expression level of transcripts whichshow standard deviation greater than 0.2 among all samples. Forhierarchical clustering, we used Pearson correlation for average linkageclustering. Ingenuity Pathway Analysis (IPA) tool was used to identifythe enriched pathways. Only those pathways with the number of genes >5were selected.

Cardiac differentiation of human ESCs and iPSCs. Human ESCs and iPSCswere cultured on Matrigel (BD Biosciences)-coated surface with mTESR-1human pluripotent stem cell culture medium (STEMCELL Technologies) to80% confluence. On day 0, cells were dissociated with Accutase (Sigma)to small clumps containing 10-20 cells and resuspended in 2 ml basicmedia (StemPro34, Invitrogen, containing 2 mM glutamine, Invitrogen, 0.4mM monothioglycerol, Sigma, 50 μg/ml ascorbic acid, Sigma, and 0.5ng/ml⁻ BMP4, R&D Systems) to form embryoid bodies (EBs). On day 1-4,BMP4 (10 ng/ml), human bFGF (5 ng/ml), and activin A (3 ng/ml) wereadded to the basic media for cardiac specification. On day 4-8, EBs wererefreshed with basic media containing human DKK1(50 ng/ml) and humanVEGF (10 ng/ml), followed by basic media containing human bFGF (5 ng/ml)and human VEGF (10 ng/ml) starting day 8. All factors were obtained fromR&D Systems. Cultures were maintained in a 5% CO₂/air environment.

Microelectrode array (MEA) recordings. One to six beating iPSC-CM EBswere plated 1-3 days prior to experiments at day 19-47 postdifferentiation on gelatin coated MEA probes (Alpha Med Scientific).Signals were acquired at 20 kHz with a MED64 amplifier (Alpha MedScientific) and digitized using National Instruments A/D cards and a PCwith MED64 Mobius QT software. Field potential duration (FPD) wasmeasured and determined as described, and corrected offline using IGORPro (Lake Oswego) and MS Excel. FPD was normalized to the beat frequencyusing the Bazzet correction formula: cFPD=FPD/AlInterspike interval.Statistical analyses comparing the DCM and control iPSC-CMelectrophysiological parameters were performed using two tailedStudent's t-tests.

Patch clamping. Dissociated iPSC-CMs were seeded on gelatin-coated 15 mmround coverslips in 24-well plates for experiments. Whole-cell patchclamp recordings in CMs generated from control and DCM iPSCs oncoverslips were conducted using EPC-10 patch-clamp amplifier (HEKA) andan inverted microscope (Nikon, TE2000-U). Glass pipettes were preparedusing borosilicate glasses with a filament (Sutter Instrument,#BF150-110-10) using the following parameters (Heat, Velocity, Time): 1)740, 20, 250; 2) 730, 20, 250; 3) 730, 20, 250; 4) 710, 47, 250, using amicropipette puller (Sutter Instrument, Model P-87). Recordings wereconducted using the following pipette solution: 120 mM K D-gluconate, 25mM KCl, 4 mM MgATP, 2 mM NaGTP, 4 mM Na2-phospho-creatin, 10 mM EGTA, 1mM CaCl2 and 10 mM HEPES (pH 7.4 with KCl at 25° C.) in Tyrodes solution(140 mM NaCl, 5.4 mM KCl, 1 mM MgCl₂, 10 mM glucose, 1.8 mM CaCl₂, and10 mM HEPES pH 7.4 with NaOH at 25° C.). Statistical analyses wereperformed using two tailed Student's t-tests.

Single cell microfluidic PCR. Single beating CMs were picked manuallyunder the microscope. Each cell was introduced into PCR tubes containing10 μl of a mixture of reaction buffer (CellsDirect kit, Invitrogen), TEbuffer (Ambion), primers of interest (Applied Biosystems) andSuperScript III Reverse Transcriptase/Platinum Taq Mix (Invitrogen).Reverse transcription and specific transcript amplification wereperformed on the thermocycler (ABI Veriti) as follows: 50° C. for 15min, 70° C. for 2 min, 94° C. for 2 min, then 94° C. for 15 sec, 60° C.for 30 sec, and 68° C. for 45 sec for 18 cycles, then 68° C. for 7minutes. The amplified cDNA was loaded into Biomark 48.48 Dynamic Arraychips using the Nanoflex IFC controller (Fluidigm). Threshold cycle (CT)as a measurement of relative fluorescence intensity was extracted by theBioMark Real-Time PCR Analysis software (Fluidigm).

Endothelial cell differentiation. iPSCs were dispersed into cellaggregates containing approximately 500 to 1,000 cells using 1 mg/mlcollagenase IV (Invitrogen). Cell aggregates were suspension cultured inultra-low attachment cell culture dishes in Knockout DMEM containing 20%ES-Qualified FBS (Invitrogen) supplemented with inductive cytokines (R&DSystems) as follows: day 0-7: 20 ng/ml BMP4; day 1-4: 10 ng/ml ActivinA; day 2-14: 8 ng/ml FGF-2; day 4-14: 25 ng/ml VEGF-A. Endothelialprogenitor cells were magnetically separated using mouse anti-human CD31antibody (BD Biosciences) and expanded in EGM-2 endothelial cell culturemedium (Lonza).

TABLE 1 Genetic screening of the DCM gene panel by next generationsequencing (Illumina) Gene Symbol Protein Coded NCBI Ref Gene No.Mutation(s) LMNA lamin A/C NG_008692 None MYH7 beta-myosin heavy chainNG_007884 None TNNT2 cardiac muscle troponin T NG_007556 p.Arg173TrpACTC1 alpha-cardiac actin NG_007553 None DES desmin NG_008043 NoneMYBPC3 cardiac myosin-binding protein C NG_007667 None TPM1 alphatropomyosin NG_007557 None TNNI3 cardiac muscle troponin I NG_007866None LDB3 LIM domain binding 3 (ZASP) NG_008876 None TAZ tafazzinNG_009634 None PLN phospholamban NG_009082 None TTR transthyretinNG_009490 None LAMP2 lysosomal-associated membrane protein 2 NG_007995None SGCD delta sarcoglycan NG_008693 None MTTL1 mitochondrially encodedtRNA leucine 1 NC_012920_TRNL1 None MTTQ mitochondrially encoded tRNAglutamine NC_012920_TRNQ None MTTH mitochondrially encoded tRNAhistidine NC_012920_TRNH None MTTK mitochondrially encoded tRNA lysineNC_012920_TRNK None MTTS1 mitochondrially encoded tRNA serine 1NC_012920_TRNS1 None MTTS2 mitochondrially encoded tRNA serine 1NC_012920_TRNS2 None MTND1 mitochondrially encoded NADH NC_012920_ND1None dehydrogenase 1 MTND5 mitochondrially encoded NADH NC_012920_ND5None dehydrogenase 5 MTND6 mitochondrially encoded NADH NC_012920_ND6None dehydrogenase 6

TABLE 2 Clinical characteristics of the R173W DCM family Genotype RVSize LA Size Pedigree Age Clinical TNNT2 LVEDD Ejection in Diastole inSystole ID (yrs) Diagnosis (p.Arg173Trp) (cm) Fraction (%) (cm) (cm) Ia75 DCM Arg173Trp 6.0 25-30% 2.7 4.2 Ib 77 — Reference NA NA NA NA IIa 46DCM Arg173Trp 5.6 35%(2006), WNL WNL 50%(2011)  IIb 50 DCM Arg173Trp WNL40-45%  3.32 WNL IIc 48 — Reference 5.3 65-70% WNL 4.0 IIIa 16 DCMArg173Trp 6.7   19% 1.8 4.7 IIIb 18 — Reference WNL   58% 2.9 3.6 LVEDD,Left Ventricular End Diastolic Diameter Normal LVEDD Range (Adult), <5.5cm Normal ejection fraction (EF), >55% WNL = Within Normal Limits Normalright ventricle (RV) diastole size = <3.8 cm Normal left atrium (LA)size systole = <4.2 cm

TABLE 3 Exome sequencing and screening of 32 recently updated list ofgenes causing DCM for patient IIIa did not uncover additional singlenucleotide variants which could potentially account for the diseasephenotype Whole exome SNVs called* 49,143 Number of called SNVs indbSNP132 45,501 Among 32 DCM genes (MYH6, MYH7, MYPN, TNNT2, SCN5A,MYBPC3, RBM20, TMPO, LAMA4, VCL, LDB3, TCAP, PSEN1, PSEN2, ACTN2, CRYAB,TPM1, ABCC9, ACTC1, PDLIM3, ILK, TNNC1, TNNI3, PLN, DES, SGCD, CSRP3,TTN, EYA4, ANKRD1, DMD, TAZ) SNVs called    83 Number of called SNVs indbSNP132    78 Non-synonymous SNVs    37 Deleterious by SIFT    24Deleterious and absent in dbSNP132 chr1: 201332477 C−>T, TNNT2 R173Wchr2: 179634421 T−>G, TTN T2917P chr2: 179422181 C−>T, TTN V20205I chr2:179398195 C−>G, TTN E25318Q chr1: 201332477 C−>T, TNNT2 R173W Segregatewith DCM and verified by genomic PCR and DNA sequencing chr2: 179634421T−>G, TTN T2917P Exome sequencing error verified by genomic PCR and DNAsequencing chr2: 179422181 C−>T, TTN V20205I Not segregate with DCM andverified by genomic PCR and DNA sequencing chr2: 179398195 C−>G, TTNE25318Q Not segregate with DCM and verified by genomic PCR and DNAsequencing *Using default GATK filter parameters for PASS SNVs, singlenucleotide variants

TABLE 4 Baseline electrophysiological parameters of iPSC-derived beatingEBs obtained via MEA Recordings Corrected Field Field PotentialPotential Minimum Maximum Beats Per Duration Duration AmplitudeAmplitude iPSC-EBs Minute (ms) (ms) (uV) (uV) Control 70.29 ± 2.74466.56 ± 18.15 494.00 ± 17.70 −325.90 ± 82.01 189.82 ± 39.54 (n = 45)DCM 64.88 ± 3.25 471.89 ± 20.81 470.66 ± 19.70 −199.67 ± 33.79 106.20 ±14.64 (n = 57) Mean ± s.e.m.

TABLE 5 Parameters of single DCM iPSC-CMs measured by AFM Cell TypeFrequency (sec) Force (nN) Beat Duration (sec) Control (n = 13) 1.01 ±0.28 3.56 ± 0.97 0.34 ± 0.06 DCM/DCM-Ad.GFP 0.76 ± 0.09 0.65 ± 0.05 0.37± 0.07 (n = 17) DCM-Ad.Serca2a 1.09 ± 0.17 4.35 ± 1.01 0.16 ± 0.02 (n =12) Mean ± s.e.m.

TABLE 6 Primers used for real time quantitative-PCR and allelic-PCRAmplicon Forward Primer Reverse Primer ACTB TGAAGTGTGACGTGGACATCGGAGGAGCAATGATCTTGAT (SEQ ID NO: 1) (SEQ ID NO: 2) OCT4 TotalAGCGAACCAGTATCGAGAAC TTACAGAACCACACTCGGAC (SEQ ID NO: 3) (SEQ ID NO: 4)OCT4 CCTCACTTCACTGCACTGTA CAG GTTTTCTTTCCCTAGCT Endogenous(SEQ ID NO: 5) (SEQ ID NO: 6) SOX2 Total AGCTACAGCATGATGCAGGAGGTCATGGAGTTGTACTGCA (SEQ ID NO: 7) (SEQ ID NO: 8) Sox2 EndogenousCCCAGCAGACTTCACATGT CCTCCCATTTCCCTCGTTTT (SEQ ID NO: 9) (SEQ ID NO: 10)Klf4 Total TCTCAAGGCACACCTGCGAA TAGTGCCTGGTCAGTTCATC (SEQ ID NO: 11)(SEQ ID NO: 12) Klf4 Endogenous GATGAACTGACCAGGCACTAGTGGGTCATATCCACTGTCT (SEQ ID NO: 13) (SEQ ID NO: 14) C-MYC TotalACTCTGAGGAGGAACAAGAA TGGAGACGTGGCACCTCTT (SEQ ID NO: 15) (SEQ ID NO: 16)C-MYC TGCCTCAAATTGGACTTTGG GATTGAAATTCTGTGTAACTGC Endogenous(SEQ ID NO: 17) (SEQ ID NO: 18) Nanog Total TGAACCTCAGCTACAAACAGTGGTGGTAGGAAGAGTAAAG (SEQ ID NO: 19) (SEQ ID NO: 20) TNNT2 WtGGAGGAGGAGCTCGTTTCTCTCA CATGTTGGACAAAGCCTTCTTCTT AAG (SEQ ID NO: 21)CCG (SEQ ID NO: 22) TNNT2 mutant GGAGGAGGAGCTCGTTTCTCTCACATGTTGGACAAAGCCTTCTTCTT AAG (SEQ ID NO: 23) CCA (SEQ ID NO: 24)

TABLE 7 Selected enriched pathways for rescued genes after Serca2aover-expression in DCM iPSC-CMs Canonical Pathways Genes FactorsPromoting SMAD2, BMP4, LEF1, DKK1, BMP10, PDGFRB Cardiogenesis inVertebrates Calcium Signaling HDAC9, NFATC1, CASQ2, ACTA1. TMEM38B,NECAB1, ATP1A3, ATP2A2 Integrin Signaling/Cytoskeletal TSPAN7, ITGA8,TSPAN2, ACTA1, ITGB3, ACTA1, ITGB3, KIF1A, MAP2, DYNLT3, ITGA8 HumanEmbryonic Stem Cell SMAD2, BMP4, LEF1, BMP10, PDGFRB PluripotencyProtein Ubiquitination Pathway UBD, DNAJB4, USP17, DNAJB9, HSPA4L,USP17L2, USP17L6P, UGT2B7, UHRF1, UIMC1 Wnt/β-catenin Signaling UBD,GNA01, LEF1, DKK1, SOX5 G-Protein Coupled Receptor Signaling GPR124,NPY1R, FSHR, GNA01, VN1R1, BDKRB1, OR4F16, OR4F17, OR52N5, OR5R1 ProteinKinase A Signaling LEF1, NFATC1, HIST1H1B, HIST2H3C, HIST1H3F, HIST1H3J,HIST2H3A, HIST2H3D

TABLE 8 Spread of iPSC lines analyzed by each assay Phenotype DCM CONIndividual Ia IIa IIb IIIa Ib IIc IIb Lines/Clones 1 2 1 2 1 3 1 3 1 4 23 1 2 Teratoma assay x x x x x x x x x x x x x x Karyotyping x x x x x xx x x x x x x x Bisulphite x x x x x x x x x x x x x x Beating EBs MEA xx x x x x x x x x x baseline Patch clamping x x x x x x x x x x singleCMs MEA NE treatment x x x x x x beating EBs Calcium imaging x x x x x xx x x x x x single CMs CMs sarcomeric x x x x x x x x x x x x x xintegrity analysis Sarcomeric integrity x x x x x x after NE treatmentAFM single CMs x x x x Metoprolol treatment x x x x Serca2a Treatment xx x x Microarray Serca2a x x x x Treatment

Example 2 Cardiomyocytes from Patients with Hypertrophic Cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is an autosomal dominant disease ofthe cardiac sarcomere, and is estimated to be the most prevalenthereditary heart condition in the world. Patients with HCM exhibitabnormal thickening of the left ventricular (LV) myocardium in theabsence of increased hemodynamic burden and are at heightened risk forclinical complications such as progressive heart failure, arrhythmia,and sudden cardiac death (SCD). Molecular genetic studies from the pasttwo decades have demonstrated that HCM is caused by mutations in genesencoding for proteins in the cardiac sarcomere. While identification ofspecific mutations has defined the genetic causes of HCM, the pathwaysby which sarcomeric mutations lead to myocyte hypertrophy andventricular arrhythmia are not well understood. Efforts to elucidate themechanisms underlying development of HCM have yielded conflictingresults, paradoxically supporting models of both loss in myosin functionand gain in myosin function to explain development of the disease.Attempts to resolve these discrepancies have been hampered bydifficulties in obtaining human cardiac tissue and the inability topropagate heart samples in culture.

To circumvent these hurdles, we generated induced pluripotent stemcell-derived cardiomyocytes (iPSC-CMs) from a family of 10 individuals,half of whom carry an autosomal dominant missense mutation on exon 18 ofthe β myosin heavy chain gene (MYH7) encoding for an Arginine toHistidine substitution at amino acid position 663 (Arg663His). Thegeneration of patient-specific iPSC-CMs allows for recapitulation of HCMat the single cell level and that preclinical modeling of HCM iPSC-CMscan elucidate the mechanisms underlying development of the disease.These findings validate iPSC technology as a novel method to understandhow sarcomeric mutations cause the development of HCM and to identifynew therapeutic targets for the disease.

Recruitment of HCM family cohort and evaluation of disease genotype andphenotype. A 10 member family cohort that spanned two generations (IIand III) was recruited for isolation of dermal fibroblasts. The probandwas a 53-year old African American female patient (II-1) who presentedat the hospital with palpitations, shortness of breath, and exertionalchest pain. Results from comprehensive testing revealed concentric leftventricular hypertrophy (LVH) with prominent thickening of the inferiorseptum and inferior wall (FIG. 31A). To confirm presence of an HCMcausing mutation, the proband's genomic DNA was screened for mutationswith a panel of 18 genes associated with HCM. Nucleotide sequenceanalysis demonstrated a known familial HCM missense mutation on exon 18of the β-myosin heavy chain gene, which causes an Arginine to Histidinesubstitution at amino acid position 663 (Arg663His; FIG. 31B).Subsequent genetic evaluation of the proband's family revealed that fourof her eight children (III-1, III-2, III-3, III-8; ages 21, 18, 14, 10)carried the Arg663His mutation (FIG. 31C). The proband's familyunderwent the same comprehensive clinical evaluation, which revealedmild LVH in the two eldest carriers on echocardiography and MRI as wellas occasional premature ventricular contractions on ambulatorymonitoring. The two younger carriers (III-3 and III-8; ages 14 and 10)had not fully developed the phenotype, but did exhibit hyperdynamicfunction by echocardiography. The proband's husband (II-2; age 55) andother four children (III-4, III-5, III-6, III-7; ages 20, 16, 5 14, 13)

TABLE 9 Genes Screened Gene Symbol Protein Code NCBI Ref Gene No.Mutation(s) ACTC1 alpha-cardiac actin NG_007553 None CAV3 caveolin 3NG_008797 None GLA galactosidase alpha NG_007119 None LAMP2lysosomal-associated membrane protein 2 NG_007993 None MTTGmitochondrial transfer RNA glycine NC_012920_TRNG None MTTImitochondrial transfer RNA isoleucine NC_01296_TRNI None MTTKmitochondrial transfer RNA lysine NC_012920_TRNK None MTTQ mitochondrialtransfer RNA glutamine NC_012920_TRNQ None MYBPC3 cardiac myosin-bindingprotein C NG_007667 None MYH7 beta-myosin heavy chain NG_007884Arg663His MYL2 myosin regulatory light chain 2 NG_007554 None MYL3myosin light chain 3 NG_007555 None PRKAG2 5′-AMP-activated proteinkinase subunit gamma-2 NG_007486 None TNNC1 troponin C NG_008963 NoneTNNI3 cardiac muscle troponin I NG_007866 None TNNT2 cardiac muscletroponin T MG_007556 None TPM1 alpha tropomyosin NG_007553 None TTRTransthyretin NG_009490 None

Generation of patient-specific iPSCs and confirmation of pluripotencyPatient-specific iPSCs were generated from primary fibroblasts of all 10individuals through lentiviral infection with the reprogramming factorsOct-4, Sox-2, Klf-4 and c-Myc. A minimum of 3 distinct lines wasgenerated per patient, and assayed for pluripotency through a battery oftests. Established iPSCs exhibited positive immunostaining for the ESCmarkers SSEA-4, TRA-1-60, TRA-1-81, Oct4, Sox2, Nanog, and alkalinephosphatase, as well as protein expression for the transcription factorsOct4, Sox2, and Nanog. Quantitative bisulfite pyrosequencing andquantitative RT-PCR demonstrated hypomethylation of Nanog and Oct-4promoters, activation of endogenous pluripotency transcription factors,and silencing of lentiviral transgenes. Microarray analyses comparingwhole genome expression profiles of dermal fibroblasts, iPSCs, and humanESCs (WA09 line) further confirmed successful reprogramming of all celllines. Karyotyping demonstrated stable chromosomal integrity in all iPSClines through passage 30. Spontaneous embryoid body (EB) and teratomaformation assays yielded cellular derivatives of all three germ layersin vitro and in vivo, confirming the pluripotent nature of generatediPSCs. Restriction enzyme digestion and sequencing verified the presenceand absence of the Arg663His mutation in the MYH7 locus of HCM andcontrol iPSCs respectively.

Differentiation of patient-specific iPSCs into cardiomyocytes.Established iPSC lines from all subjects were differentiated intocardiomyocyte lineages (iPSC-CMs) using standard 3D EB differentiationprotocols. Ten to twenty days following the initiation ofdifferentiation, spontaneously contracting EBs were observed to appearunder light microscopy. Immunostaining for cardiac Troponin T indicatedbeating EBs from both control and HCM iPSC lines contained cardiomyocytepurities between 60-80%. Beating EBs were seeded on multi-electrodearray (MEA) probes for evaluation of electrophysiological properties.Both control and HCM iPSC-derived EBs exhibited comparable beatfrequencies, field potentials, and upstroke velocities at baseline. EBswere subsequently dissociated into single iPSC-CMs and plated on gelatincoated chamber slides for further analysis. Single dissociated iPSC-CMsfrom both HCM and control family members maintained spontaneouscontraction and exhibited positive staining for sarcomeric proteins suchas cardiac troponin T and myosin light chain (FIG. 31D).

iPSC-CMs carrying the Arg663His mutation recapitulate HCM phenotype invitro Following cardiac differentiation and dissociation to singlebeating cells, diseased and control-matched iPSC-CMs were characterizedin vitro for recapitulation of the HCM phenotype. Hypertrophic iPSC-CMsexhibited features of HCM such as cellular enlargement andmultinucleation beginning in the sixth week following induction ofcardiac differentiation (Arad et al., 2002). At day 40 post-induction,HCM iPSC-CMs were noticeably larger (1859+517 pixels; n=236, 4 patientlines) than control matched iPSC-CMs (1175+328 pixels; n=220, 4 patientlines) and exhibited significantly higher frequencies of multinucleation(HCM: 49.7+8.5%; n=236, 4 lines vs control: 23.0+3.7%; n=220, 4 lines)(FIG. 31D-F). Mutant iPSC-CMs also demonstrated other hallmarks of HCMincluding expression of atrial natriuretic factor (ANF), elevation ofβ-myosin/α-myosin ratio, calcineurin activation, and nucleartranslocation of nuclear factor of activated T-cells (NFAT) as detectedby immunostaining (FIG. 31G-K). As calcineurin-NFAT signaling is a keytranscriptional activator for induction of hypertrophy in adultcardiomyocytes, we sought to test the importance of this pathway tohypertrophic development in HCM iPSC-CMs. Blockade of calcineurin-NFATinteraction in HCM iPSC-CMs by cyclosporin A (CsA) and FK506 reducedhypertrophy by over 40% (FIG. 31L). In the absence of inhibition,NFAT-activated mediators of hypertrophy such as GATA4 and MEF2C werefound to be significantly upregulated in HCM iPSC-CMs beginning day 40post-induction of cardiac differentiation, but not prior to this point.Taken together, these results indicate that calcineurin-NFAT signalingplays a central role in the development of the HCM phenotype as causedby the Arg663His mutation.

Single cell gene expression profiling demonstrates activation of HCMassociated genes Clinical presentation of HCM typically occurs over thecourse of several decades in affected individuals. To investigate thetemporal effects of the Arg663His mutation upon HCM development at thecellular level, we assessed the expression of hypertrophic-related genesin single purified iPSC-CMs from both HCM and control patients. Singlecontracting cardiomyocytes were manually lifted from culture dishes atdays 20, 30, and 40 from initiation of differentiation and subjected tosingle cell quantitative PCR analysis using a panel of 32cardiomyocyte-related transcripts. Beginning at day 40, hypertrophicrelated genes such as GATA4, TNNT2, MYL2, and MYH7 were found to beupregulated in HCM iPSC-CMs (FIG. 31M). No significant increases inexpression of HCM related genes were found prior to this time point.

iPSC-CMs carrying the Arg663His mutation exhibit electrophysiologicaland contractile arrhythmia at the single cell level Arrhythmia is aclinical hallmark of HCM, and is responsible for a significant portionof morbidity and mortality associated with the disease including suddencardiac death. We therefore next examined the electrophysiologicalproperties of iPSCCMs carrying the Arg663His mutation by whole cellpatch clamping. Both HCM and control iPSC-CMs contained myocytepopulations characterized by nodal-like, ventricular-like, andatrial-like electrical waveforms. In the first four weeks followinginduction of differentiation, cells from both groups displayed similaraction potential frequencies, peak amplitudes, and resting potentials.However, starting at day 30, a large subfraction (40.4+12.9%; n=131, 5patient lines) of HCM myocytes as compared to controls (5.1+7.1%; n=144,5 patient lines) were observed to exhibit arrhythmic waveforms includingfrequent small depolarizations that resembled delayedafterdepolarizations (DADs) that failed to trigger action potentials andclustered beats (FIG. 32A1, 32A2, 32B-C). Time-lapse videos of singlebeating iPSC-CMs under light microscopy confirmed thatelectrophysiological deficiencies resulted in contractile arrhythmia.Compared to control iPSCCMs (1.4+1.9%; n=68, 5 patient lines), which hadregular beat intervals, HCM iPSC-CMs contained numerous cells(12.4+5.0%; n=64, 5 patient lines) that beat at irregular frequencies.Analysis of single cell video recordings by pixel quantificationsoftware confirmed the arrhythmic nature of HCM iPSC-CM contraction.Taken together, these findings demonstrate sarcomeric mutations arecapable of inducing electrophysiological and contractile arrhythmia atthe single cell level.

Overexpression of the Arg663His mutation in normal hESC-CMsrecapitulates calcium handling abnormalities of HCM iPSC-CMs Calcium(Ca²⁺) plays a fundamental role in regulation of excitation-contractioncoupling and electrophysiological signaling in the heart. To investigatethe possible mechanisms underlying arrhythmia in myocytes carrying theArg663His mutation, we next analyzed Ca²⁺ handling properties ofiPSC-CMs from control and HCM patients using the fluorescent Ca²⁺ dyeFluo-4 acetoxymethyl ester (AM). Compared to iPSC-CMs derived fromhealthy individuals, HCM iPSC-CMs demonstrated significant Ca²⁺transient irregularities such as multiple events possibly related totriggered arrhythmia-like voltage waveforms, which were virtually absentin control cells (FIG. 32D-E). Interestingly, irregular Ca₂₊ transientswere observed to occur in HCM iPSC-CMs prior to the onset of cellularhypertrophy, suggesting that abnormal Ca²⁺ handling may be a causalfactor for the induction of the hypertrophic phenotype. Becausevariations inherent to spontaneous contractions can potentially confoundCa²⁺ transients, we subjected HCM and control iPSC-CMs to 1 Hz pacingduring line scanning. Consistent with data from spontaneous contraction,abnormal Ca₂₊ transients were found to be common in HCM iPSC-CMs(12.5+4.9%; n=19, 5 patient lines) and absent in control iPSC-CMs (n=20,5 patient lines). To further ensure that observed deficiencies inelectrophysiology and Ca²⁺ regulation were due to the Arg663Hismutation, we next over-expressed the mutant form of myosin in humanembryonic stem cell-derived cardiomyocytes (hESC-CMs; WA09 line).hESC-CMs overexpressing the Arg663His mutant MYH7 transcript were foundto exhibit similar arrhythmias and abnormal Ca²⁺ transients (FIG.32F-I).

Previous reports have linked intracellular Ca²⁺ ([Ca²⁺]_(i)) elevationas a trigger for arrhythmia and cellular hypertrophy. We thereforefurther compared [Ca²⁺]_(i) in control and diseased iPSC-CMs.Preliminary quantification of [Ca²⁺]_(i) by Fluo-4 baseline intensitysuggested that [Ca²⁺]_(i) was approximately 30% higher in HCM iPSCCMs(n=105, 4 patient lines) than control counterparts (n=122, 4 patientlines) at day 30 postinduction (FIG. 32J). To confirm diastolic[Ca²⁺]_(i) differences, we also used the ratiometric Ca²⁺ dye Indo-1 incontrol and HCM iPSC-CMs. Indo-1 imaging demonstrated that diastolic[Ca²⁺]_(i) was higher (25.1% increase in Indo-1 ratio) in iPSC-CMscarrying the Arg663His mutation (n=26, 4 patient lines) as compared tocontrol cells (n=17, 4 patient lines), and that arrhythmic activity wasapparent in only the Arg663His myocytes. These findings emphasize a rolefor irregular Ca²⁺ cycling in the pathogenesis of HCM (FIG. 32K-L).

Measurement of sarcoplasmic reticulum (SR) Ca²⁺ stores further supportedfindings of elevated [Ca₂₊]_(i) in diseased iPSC-CMs as cytoplasmicretention of Ca²⁺ has been shown to decrease SR Ca²⁺ load by impeding SRCa²⁺ uptake. HCM and control iPSC-CMs were loaded with Fluo-4 andexposed to caffeine, which induces release of SR Ca₂₊ stores into thecytoplasm. Myocytes carrying the Arg663His mutation were characterizedby significantly smaller SR Ca²⁺ release (mean peak ΔF/F0ratio=3.05+0.20, n=35, 3 patient lines) as compared to control iPSC-CMs(mean peak ΔF/F0 ratio=3.96+0.18, n=23, 3 patient lines) (FIG. 32M-N).These findings demonstrate a central role for Ca₂₊ cycling dysfunctionand elevated [Ca²⁺]_(i) in the pathogenesis of HCM as caused by theArg663His mutation.

Inotropic stimulation exacerbates HCM phenotype in diseased iPSC-CMsBecause iPSC-CMs carrying the Arg663His mutation recapitulated numerousaspects of the HCM phenotype in vitro, we hypothesized that our platformcould also be used as a screening tool to assess the effect ofpharmaceutical drugs upon HCM at the single cell level. To test thecapacity of HCM iPSC-CMs to accurately model pharmaceutical drugresponse, we first subjected single control and diseased iPSC-CMs topositive inotropic stimulation, a known trigger for myocyte hypertrophyand ventricular tachycardia. Patient-specific cardiomyocytes wereincubated β-adrenergic agonist (200 μM isoproterenol) on a daily basisfor 5 days beginning 30 days after induction of differentiation.Previously HCM iPSC-CMs typically did not exhibit cellular hypertrophyuntil day 40 post-induction (FIG. 31E), but were found to increase incell size by 1.7-fold between day 30 and 35 as compared to controlcounterparts when treated with isoproterenol (FIG. 33A). β-adrenergicstimulation was also found to severely exacerbate presentation ofirregular Ca₂₊ transients and arrhythmia in HCM iPSC-CMs (FIG. 33B-C).Importantly, co-administration of β-adrenergic blocker (400 μMpropranolol) with isoproterenol significantly amelioratedcatecholamine-induced exacerbation of hypertrophy, Ca²⁺ handlingdeficiencies, and arrhythmia.

Treatment of Ca²⁺ dysregulation prevents development of the HCMphenotype We therefore assessed whether pharmaceutical inhibition ofCa²⁺ entry could help prevent HCM phenotype development by treatingcontrol and mutant iPSC-CMs with the L-type Ca²⁺ channel blockerverapamil. Compared to control cells, the spontaneous beating rate inHCM iPSC-CMs was relatively resistant to verapamil as detected by MEAdose-response experiments (HCM IC₅₀=930.61+80.0 nM, controlIC₅₀=103.0+6.0 nM), consistent with the elevated [Ca²⁺]_(i) in iPSC-CMscarrying the Arg663His mutation. Remarkably, continuous addition ofverapamil at therapeutic dosages (50-100 μM) to single diseased iPSC-CMsfor 10-20 sequential days significantly ameliorated all aspects of theHCM phenotype including myocyte hypertrophy, Ca²⁺ handlingabnormalities, and arrhythmia (FIG. 34A-C).

Arrhythmic iPSC-CMs can be screened for potential pharmaceuticaltreatments at the single cell level As current pharmaceutical therapyfor HCM includes the use of β-blockers and antiarrhythmics in additionto Ca²⁺ channel blockers we further screened a panel of 12 other drugsused clinically to treat HCM for their potential to ameliorate the HCMphenotype at the single cell level. While verapamil was the only agentfound to be capable of preventing cellular hypertrophy, anti-arrhythmicdrugs which inhibit Na₊ influx such as lidocaine, mexiletine, andranolazine also demonstrated potential to restore normal beat frequencyin HCM iPSC-CMs, possibly through limiting Ca²⁺ entry into the cell bythe Na₊/Ca₂₊ exchanger. Other anti-arrhythmic agents targeting K₊channels and β-blockers administered in the absence of inotropicstimulation did not have any therapeutic effects in single cells.Altogether, these results implicate imbalances in Ca₂₊ regulation as acentral mechanism underlying development of HCM at the cellular leveland demonstrate the potential of patient-specific iPSC-CMs as a powerfultool for the identification of novel pharmaceutical agents to treat HCM.

TABLE 10 Drugs that were Screened Video Therapeutic Concentrations DrugClass Target Analysis Effect Tested Quinidine Ia Na⁺ channel blocker Xno 0.1-20 uM (intermediate association/dissociation) Procainamide Ia Na⁺channel blocker ⊚ no 1-200 uM (intermediate association/dissociation)Lidocaine Ib Na⁺ channel blocker (fast ⊚ yes 1-100 nMassociation/dissociation) Mexiletine Ib Na⁺ channel blocker (fast ⊚ yes1-50 uM association/dissociation) Ranolazine NA Late Na⁺ channel blocker⊚ yes 0.1-10 uM Flecainide Ic Na⁺ channel blocker (slow ⊚ no 0.1-5 uMassociation/dissociation Propafenone Ic Na⁺ channel blocker (slow ⊚ no1-100 uM association/dissociation Propranolol II Beta-blocker ⊚ no 1-400uM Metoprolol II Beta-blocker X no 0.1-20 uM Amiodarone II K⁺ channelblocker X no 0.1-10 uM Sotalol III K⁺ channel blocker X no 1-400 uMDofetilide III K⁺ channel blocker X no 0.1-20 uM Verapamil IV Ca²⁺channel blocker X yes* 1-100 uM *Verapamil was only observed to have atherapeutic effect upon HCM iPSC-CMs following continuous addition tothe culture media for 5 or more days in a row. Treatment for a period oftime less than 5 days was not observed to have any therapeutic effectsupon Ca²⁺ handling or arrhythmogenicity. All other durg screening assayswere conducted by incubating cells with respective pharmaceuticalcompunds at the listed concentrations for 10 minutes followed bywashout.The genetic causes of HCM were initially identified several decades ago.However, the mechanisms by which mutations in genes encoding for thecardiac sarcomere can cause development of HCM remain unclear.Generation of patient-specific iPSC-CMs has allowed for in depthmodeling of hereditary cardiovascular disorders including dilatedcardiomyopathy, LEOPARD and long QT syndrome. To elucidate the signalingpathways underlying HCM development, we utilized iPSC technology togenerate functional cardiomyocytes from dermal fibroblasts of a10-member family cohort, half of whom possess the HCM Arg663His mutationin the MYH7 gene. Patient-specific iPSC-CMs recapitulated a number ofcharacteristics of HCM including cellular hypertrophy, calcineurin-NFATactivation, upregulation of hypertrophic transcription factors, andcontractile arrhythmia. Irregular Ca²⁺ transients and elevation ofdiastolic [Ca²⁺]_(i) were observed to precede the presentation of otherphenotypic abnormalities, strongly implicating dysregulation of Ca²⁺cycling as a central mechanism for pathogenesis of the disease.Imbalances in Ca²⁺ homeostasis have been described as a keycharacteristic of HCM in numerous reports. However, little evidenceexists to delineate whether these abnormalities are a symptom of HCM ora causal factor.

In this study, we present several lines of evidence for a crucial roleof Ca²⁺ in development of HCM as caused by the Arg663His mutation.Specifically, our findings show that an elevation in [Ca²⁺]_(i) mediatedby the Arg663His mutation can induce both cellular hypertrophy andcontractile arrhythmia. The sustained elevation of [Ca²⁺]μs a knowntrigger for activation of calcineurin, a Ca²⁺ dependent phosphatase thatis a critical effector of hypertrophy in myocytes under conditions ofstress. Activated calcineurin dephosphorylates NFAT3 transcriptionfactors, allowing their translocation to the nucleus for directinteraction with classical mediators of hypertrophy such as GATA4 andMEF2. Time-based gene expression profiling of single iPSC-CMs followinginduction of cardiac differentiation confirmed this model as expressionof downstream effectors of hypertrophy was observed to be dependent on[Ca²⁺]_(i) elevation and nuclear translocation of NFAT. Inhibition ofcalcineurin activity by CsA and FK506 as well as reduction of Ca²⁺influx by verapamil mitigated cellular hypertrophy, confirming the roleof Ca²⁺ dysfunction and calcineurin-NFAT signaling in HCM pathogenesis(FIG. 34D). Alterations in Ca²⁺ cycling are a common trigger for cardiacarrhythmias, which are a serious clinical complication of HCM due totheir potential to induce stroke or sudden cardiac death.

The mechanisms underlying arrhythmia in patients with HCM are not wellunderstood, although reports have implicated interstitial fibrosis,abnormal cardiac anatomy, myocyte disarray, increased cell size, anddysfunction in Ca²⁺ homeostasis as possible mediators. Our findingsdemonstrate for the first time that the Arg663His mutation in the MYH7gene can directly result in electrophysiological and contractilearrhythmia at the single cell level even in the absence of cellularhypertrophy. The most likely mechanism for development of arrhythmia inindividual HCM iPSC-CMs is buildup of [Ca²⁺]_(i), which induces delayedafter depolarizations (DADs) whereby sarcoplasmic reticulum Ca²⁺ releasetriggers transient inward current following action potentialrepolarization. Continued presentation of DADs can in turn lead toventricular tachycardia and sudden cardiac death, as in patientssuffering from recurrent arrhythmia.

Whole cell current clamp experiments of HCM iPSC-CMs supported thishypothesis through demonstration of frequent spontaneous DAD-likewaveforms in diseased myocytes. We believe these results are the firstreport to demonstrate that specific HCM mutations such as Arg663His canact as direct triggers for arrhythmia at the single cell level.

The mechanistic role of elevated myocyte Ca²⁺ loading seems to becentral to both hypertrophy and arrhythmogenesis. Pharmaceutical drugscreening of mutant iPSC-CMs further supported elevated [Ca²⁺]_(i) as acentral mechanism for arrhythmia development. Of the 13 agents we used,only pharmaceutical blockade of Ca²⁺ and Na⁺ entry mitigated contractilearrhythmia in HCM iPSC-CMs. Reduction of Na⁺ influx limits [Ca²⁺]_(i) byallowing Na⁺/Ca²⁺ exchange to remove Ca²⁺ more readily. Our resultsdemonstrate the utility of iPSC-based technology to model development ofHCM and associated triggered arrhythmias, as well as to identifypotential therapeutic agents for the disease. These results are thefirst to provide direct evidence of imbalances in Ca²⁺ homeostasis as aninitiating factor in the development of HCM at the single cell level.

Experimental Procedures

Patient recruitment. Clinical evaluation of the proband and familyincluded physical examination, ECG, cardiac magnetic resonance imaging(MRI), and 24-hour Holter monitoring. Results revealed hyperdynamicventricular systolic function with near complete obliteration of theapical walls at end systole in the proband (II-1) and the eldest twocarriers (III-1 and III-2). No delayed enhancement was found on contrastenhanced MRI in the proband or carriers. Ambulatory monitoring revealedoccasional premature ventricular contractions. The youngest two carriers(III-3 and III-8; ages 14 and 10) exhibited hyperdynamic cardiacfunction but no other clinical features of HCM most likely due to theiryoung age.

Isolation and maintenance of fibroblast cells. Freshly isolated skinbiopsies were rinsed with PBS and transferred into a 1.5 ml tube. Tissuewas minced in collagenase I (1 mg/ml in Dulbecco's modified Eagle medium(DMEM), Invitrogen, Carlsbad, Calif.) and allowed to digest for 6 hoursat 37° C. Dissociated dermal fibroblasts were plated and maintained withDMEM containing 10% FBS (Invitrogen), Glutamax (Invitrogen), 4.5 g/Lglucose (Invitrogen), 110 mg/L sodium pyruvate (Invitrogen), 50 U/mLpenicillin (Invitrogen), and 50 g/mL streptomycin (Invitrogen) at 37°C., 95% air, and 5% CO2 in a humidified incubator. All cells were usedfor reprogramming within five passages.

Lentivirus production and transduction. 293FT cells (Invitrogen) wereplated at 80% confluency on 100-mm dishes and transfected with 12 μg ofthe lentiviral vectors (Oct4, Sox2, Klf4, and c-MYC) plus 8 μg ofpackaging pPAX2 and 4 μg of VSVG plasmids using Lipofectamine 2000(Invitrogen) following the manufacturer's instructions. Supernatant wascollected 48 h after transfection, filtered through a 0.45-μm pore-sizecellulose acetate filter (Millipore, Billerica, Mass.), and mixed withPEG-it Virus Concentration Solution (System Biosciences, Mountain View,Calif.) overnight at 4° C. Viruses were precipitated at 1,500 g the nextday and resuspended with Opti-MEM medium (Invitrogen).

Derivation of patient-specific iPSCs. Generation, maintenance, andcharacterization of patient-specific iPSC lines were performed aspreviously described using lentivirus as produced above onMatrigel-coated tissue culture dishes (BD Biosciences, San Jose, Calif.)with mTESR-1 hESC Growth Medium (StemCell Technology, Vancouver, Canada)

Alkaline phosphatase staining. Alkaline phosphatase (AP) staining wasconducted as in previous studies using the Quantitative AlkalinePhosphatase ES Characterization KitS (Millipore) using themanufacturer's instructions.

Immunofluorescence staining. Immunofluorescent stains were performedusing the following primary antibodies: SSEA-3, SSEA-4, Tra-1-60,Tra-1-81, ANF, Tuj-1 (Millipore), Oct3/4, Nanog, AFP (Santa Cruz,Calif.), Sox2 (Biolegend, San Diego, Calif.), smooth muscle actin(Biolegend), sarcomeric α-actinin (Sigma, St. Louis, Mo.), (cTnT (ThermoScientific Barrington, Ill.), Alexa Fluor 488 Phalloidin (invitrogen),Myosin light chain 2a (MLC2a), Myosin light chain 2v (MLC2v) (SynapticSystems, Goettingen, Germnay), and AlexaFluor conjugated secondaryantibodies (Invitrogen) as previously described.

Bisulphite pyrosequencing. The Zymo DNA Methylation Kit (Zymo Research,Irvine, Calif.) was used to treat 1 μg of sample DNA with bisulfite asper the manufacturer's instructions. Following PCR, cDNA was convertedto single-stranded DNA templates and sequenced by a Pyrosequencing PSQ96HS System (Biotage, Charlotte, N.C.). QCpG software (Biotage) was usedto analyze each individual locus as a T/C SNP.

Microarray hybridization and data analysis. RNA was isolated from iPSCsand hybridized to a Affymetrix GeneChip Human Gene 1.0 ST Array(Affymetrix, Santa Clara, Calif.). Expression was normalized andannotated by the Affymetrix Expression Console software (Affymetrix).The Pearson Correlation Coefficient was calculated for each pair ofsamples using the expression level of transcripts which shows standarddeviation greater than 0.2 among all samples.

Spontaneous in vitro differentiation. For embroid body (EB) formation,iPSC colonies were dissociated on Matrigel coated plates withcollagenase IV (Invitrogen), and seeded into low attachment 6-wellplates in Knockout DMEM (Invitrogen) containing 15% KSR (Invitrogen),Glutamax (Invitrogen), 4.5 g/L glucose (Invitrogen), 110 mg/L sodiumpyruvate (Invitrogen), 50 U/mL penicillin (Invitrogen), and 50 g/mLstreptomycin (Invitrogen) to form embroid bodies (EBs). After 5 days,EBs were transferred to adherent, gelatin-coated chamber slides andcultured in the same medium for another 8 days.

Teratoma formation. 1×10⁶ undifferentiated iPSCs were suspended in 10 μLMatrigel (BD Biosciences) and delivered by a 28.5 gauge syringe to thesubrenal capsule of 8 week old SCID Beige mice. Eight weeks after celldelivery, tumors were explanted for hematoxylin and eosin staining.

Western blot. Whole cell extracts were isolated using RIPA buffer and 10μg protein was analyzed by Western blot using specific antibodiesagainst Oct4, c-Myc, Klf4, Actin (Santa Cruz), Sox2 (Biolegend).

Cardiac differentiation of human ESCs and iPSCs. Human H9 ESCs and iPSCswere differentiated into cardiomyocytes as previously described.Briefly, pluripotent stem cells were dissociated with accutase (Sigma)at 80% confluence into small clumps of 10-20 cells. Cells wereresuspended in 2 ml basic media containing StemPro34 (Invitrogen), 2 mMglutamine (Invitrogen), 0.4 mM monothioglycerol (Sigma), 50 μg/mlascorbic acid (Sigma), and 0.5 ng/ml BMP4 (R&D Systems, Minneapolis,Minn.) to form EBs. For days 1-4 of cardiac differentiation, cells weretreated with 10 ng/ml BMP4, 5 ng/ml human bFGF (R&D Systems), and 3ng/ml activin A (R&D Systems) added to the basic media. From days 4-8,EBs were refreshed with basic media containing human 50 ng/ml DKK1 (R&DSystems) and 10 ng/ml human VEGF (R&D Systems). From day 8 onwards,cells were treated with basic media containing 5 ng/ml human bFGF and 10ng/ml human VEGF. Cultures were maintained in a 5% CO₂/air environment.

Measurement of cardiomyocyte size. For single cell cardiomyocyteanalysis, beating EBs were plated on gelatin-coated dishes. Three daysafter plating, EBs were trypsinized, filtered through a 40-mm sizepore-size filter, and single cells re-plated at low density ongelatin-coated chamber slides (Nalgene Nunc International, Rochester,N.Y.). Three days after re-plating, cells were fixed with 4%paraformaldehyde (Sigma), permeabilized in 0.3% Triton (Sigma), blockedusing 5% BSA, and stained for cardiac troponin T (1:200, Thermo Fisher)overnight at 4° C. Stained cells were washed three times with PBS, andthen incubated with the Alexa Fluor 488 phalloidin (Invitrogen), AlexaFluor 594 donkey-anti-mouse antibody (Invitrogen) and DAPI (Invitrogen)for 1 h. Cellular areas of normal and HCM iPSC-CMs were analyzed usingthe ImageJ software package (National Institutes of Health, Bethesda,Md.).

Single cell microfluidic PCR. Single beating iPSC-CMs were pickedmanually under light microscopy and placed into separate PCR tubes forreverse transcription and cDNA amplification with specified primers aspreviously described. Amplified cDNA was loaded into Biomark 48.48Dynamic Array chips (Fluidigm, South San Francisco, Calif.) for analysisby the BioMark Real-Time PCR Analysis software (Fluidigm).

Calcium (Ca²⁺) imaging. iPSC-CMs were dissociated and seeded ingelatin-coated 8-well LAB-TEK® II chambers (Nalgene Nunc International)for calcium imaging. Cells were loaded with 5 μM Fluo-4 AM (Invitrogen)and 0.02% Pluronic F-127 (Invitrogen) in Tyrodes solution (140 mM NaCl,5.4 mM KCl, 1 mM MgCl₂, 10 mM glucose, 1.8 mM CaCl², and 10 mM HEPES pH7.4 with NaOH at 25° C.) for 15 min at 37° C. Following Fluo-4 loading,cells were washed three times with Tyrodes solution. Imaging wasconducted with a confocal microscope (Carl Zeiss, LSM 510 Meta,Gottingen, Germany) with a 63× lens using Zen software (Carl Zeiss). Forpaced calcium dye imaging, fluorescence was measured at 495+20 nmexcitation and 515±20 nm emission. Videos were taken at 20 fps for 10 srecording durations. Cells were stimulated at 1 and 2 Hz. Measurementswere taken on an AxioObserver Z1 (Carl Zeiss) inverted microscopeequipped with a Lambda DG-4 300 W Xenon light source (SutterInstruments, Novato, Calif.), an ORCA-ER CCD camera (Hamamatsu,Bridgewater, N.J.), and AxioVison 4.7 software (Carl Zeiss). In eachvideo frame, regions of interest (ROls) were analyzed for changes in dyeintensity f/f0, with the resting fluorescence value f0 determined at thefirst frame of each video. Background intensity was subtracted from allvalues, and plots were normalized to zero.

Measurement of basal [Ca²⁺]_(i) using Indo-1-AM. Cardiomyocytes wereloaded in a culture medium containing 5 μM Indo-1 AM (Invitrogen) and0.02% Pluronic F-127 (Invitrogen) for 20 minutes at 37° C. After Indo-1loading, cells were washed three times with 2 mM Ca²⁺ Ringer (155 mMNaCl, 4.5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM D-glucose, and 5 mMNa-HEPES, pH 7.4) and incubated for 20 minutes at room temperature toallow for Indo-1 de-esterification. Cardiomyocytes were imaged in Ca²⁺Ringer at 32° C. using a Zeiss Axiovert 200M epifluorescence microscope.Indo-1 was excited at 350±10 nm using a 0.6 UVND filter (to attenuateexcitation intensity) and a 400 DCLP. The emitted light was separatedusing a Cairn Optosplit II (425 dichroic, 488/22 bandpass filter, Kent,UK). Spontaneous Ca²⁺ transients were collected with 4×4 pixel binningin stream acquisition mode using Metamorph software (Molecular Devices,Sunnyvale, Calif.) at 100 ms exposures. For image analysis, short andlong wavelength emission channels were aligned using the Cairn ImageSplitter ImageJ plugin.

Caffeine treatment of iPSC-CMs. Cells were perfused with PBS containing1.8 mM Ca²⁺ and 1 mM Magnesium and paced at 1 Hz to view regulartransients. A two second puff of 20 mM stock caffeine solution wasdelivered through a perfusion apparatus. Pacing was turned off prior tocaffeine reaching the cells in order to accurately measure Ca₂₊ release.

Analysis of calcium imaging linescans. Average fluorescence intensityfor Ca²⁺ linescans was quantified using Fiji (National Institutes ofHealth). Timing between transients was defined as the time between thepeaks of two successive spikes. The Ca²⁺ baseline was defined as themedian of all minima of transients. Irregularity for spike timing wasdefined as the ratio of the standard deviation (s.d.) to the mean.

Microelectrode array (MEA) recordings. Control and HCM iPSCs weredifferentiated into beating EBs ranging from 60-80% purity of CMs andseeded onto multi-electrode 40 arrays for recording of field potentialduration (FPD) and beating frequency (beats per minute, BPM) andinterspike intervals (ISI). Beating iPSC-CM EBs were plated ongelatin-coated MEA probes (Alpha Med Scientific, Osaka, Japan) prior toexperiments 20-40 days post-differentiation. Signals were acquired at 20kHz with a MED64 amplifier (Alpha Med Scientific) and digitized using aPC with PCI −6071 ND cards (National Instruments, Austin, Tex.) runningMED64 Mobius QT software (Witwerx, Inc., Tustin, Calif.). Allexperiments were performed at 35.8 to 37.5° C. in DMEM without serum orantibiotics. Stock verapamil solutions were made in double distilledwater at a 50 mM concentration. Dose-response experiments were performedby adding 0.4 to 2 μL of 1000× verapamil concentrations in DMEM to the1-2 ml volume in the MEA probe for 10 minutes at each dose. Beatingfrequencies and field potential waveform data were extracted offlineusing Mobius QT and saved as CSV files. Waveform data was imported intoIGOR Pro (Wavemetrics, Portland, Oreg.) for FPD and Vmax measurements.Beat frequencies were normalized to baseline for verapamil dose-responseexperiments and FPDs were adjusted to the beat frequency using theBazett correction formula: cFPD=FPD/√Interspike interval.

Patch clamping. Whole-cell patch-clamp recordings were conducted usingan EPC-10 patch-clamp amplifier (HEKA, Lambrecht, Germany). ContractingEBs were mechanically isolated, enzymatically dispersed into singlecells and attached to gelatincoated glass coverslips (CS-22/40, Warner,Hamden, Conn.). While recordings, the coverslips containing platedcardiomyocytes or the hERG-HEK293 cells were transferred to a RC-26Crecording chamber (Warner) mounted on to the stage of an invertedmicroscope (Nikon, Tokyo, Japan). The glass pipettes were prepared usingthin-wall borosilicate glass (Warner) using a micropipette puller(Sutter Instrument, Novato, Calif.), polished using a microforge(Narishige, Tokyo, Japan) and had resistances between 2-4 MO.Extracellular solution perfusion was continuous using a rapid solutionexchanger (Bio-logic, Grenoble, France) with solution exchange requiring1 min. Data were acquired using PatchMaster software (HEKA, Germany) anddigitized at 1.0 kHz. Data were analyzed using PulseFit (HEKA), Igor Pro(Wavemetrics, Portland, Oreg.), Origin 6.1 (Microcal, Northampton,Mass.), and Prism (Graphpad, La Jolla, Calif.). For the whole-cell patchclamp recordings of human cardiomyocytes generated from iPSCs,temperature was maintained constant by a TC-324B heating system (Warner)at 36-37° C. Current clamp recordings were conducted in normal Tyrodesolution containing 140 mM NaCl, 5.4 mM KCl, 1 mM MgCl₂, 10 mM glucose,1.8 mM CaCl₂ and 10 mM HEPES (pH 7.4 with NaOH at 25° C.). The pipettesolution contained 120 mM KCl, 1 mM MgCl₂, 10 mM HEPES, 3 mM Mg-ATP, 10mM EGTA (pH 7.2 with KOH at 25° C.). Verapamil (Sigma) was dissolved inH₂O and prepared as a 10 mM stock in a glass vial. The stock solutionwas mixed vigorously for 10 min at room temperature. For testing, thecompound was diluted in a glass vial using external solution; thedilution was prepared no longer than 30 min before using. Equal amountsof DMSO (0.1%) were present at final dilution.

Quantitative RT-PCR. Total mRNA was isolated using TRIZOL and 1 μg wasused to synthesize cDNA using the Superscript II cDNA synthesis kit(Invitrogen). 0.25 μL of the reaction mixture was used to quantify geneexpression by qPCR using SYBR® Green Master Mix (Invitrogen). Expressionvalues were normalized to the average expression of GAPDH.

Drug treatment. Single contracting iPSC-CMs were treated withpharmaceutical agents for 10 minutes for immediate analysis followed bywash out. For inotropic stimulation experiments, 200 μM isoproterenoland 400 μM propranolol were added to the cell medium for 5 continuousdays. Verapamil treatment was conducted by adding 50 μM and 100 μM tothe culture medium of iPSC-CMs for 10-20 continuous days on a dailybasis.

Example 3 Cardiomyocytes from Patients with Anthracycline Toxicity

Anthracycline-induced cardiotoxicity (and resistance toanthracycline-induced toxicity). Anthracyclines such as doxorubicin arefrontline chemotherapeutic agents that are used to treat leukemias,Hodgkin's lymphoma, and solid tumors of the breast, bladder, stomach,lung, ovaries, thyroid, and muscle, among other organs. The primary sideeffect of anthracyclines is cardiotoxicity, which results in severeheart failure for many of the recipients receiving regimens utilizingthis chemotherapeutic agent. Patient specific iPSC-cardiomyocytes(iPSC-CMs) were derived from individuals who are susceptible toanthracycline-induced cardiotoxicity as well as from individuals who arenot susceptible to anthracycline-induced cardiotoxicity.

These cells are useful to detect and titrate cardiotoxicchemotherapeutic drugs, as well as identify genes responsible forsusceptibility/resistance to anthracycline-induced cardiotoxicity. Agematched patients receiving anthracycline based chemo regimens wererecruited, and assessed whether the patients developedanthracycline-induced heart failure. Skin samples were collected fromthe patients and generated iPSC-CMs from the fibroblasts.

Methods for the isolation and maintenance of fibroblast cells;derivation of patient-specific iPSC cell lines; and cardiacdifferentiation of cells was performed as described in Example 1 orExample 2.

Example 4 Cardiomyocytes from Patients with ARVD

Arrhythmogenic right ventricular dysplasia (ARVD). ARVD is an autosomaldominant disease of cardiac desmosomes that results in arrhythmia of theright ventricle and sudden cardiac death. It is second only tohypertrophic cardiomyopathy as a leading cause for sudden cardiac deathin the young. Patient specific iPSC-cardiomyocytes (iPSC-CMs) werederived from a cohort of patients carrying a hereditary mutation forARVD as well as from family matched controls. These cell lines may beused for drug screening and to identify molecular targets responsiblefor the disease phenotype.

Methods for the isolation and maintenance of fibroblast cells;derivation of patient-specific iPSC cell lines; and cardiacdifferentiation of cells was performed as described in Example 1 orExample 2.

The iPSC-CMs were made from the blood of 6 patients. 2 patients had aP672fsX740 2013delC mutation in the PKP2 gene, 2 patients had a Q617X1849C>T mutation in the PKP2 gene, and 2 patients were family matchedcontrol subjects.

Example 5 Cardiomyocytes from Patients with LVNC

Left Ventricular Non-Compaction (LVNC, aka non-compactioncardiomyopathy). LVNC is a hereditary cardiac disease which results fromimpaired development of the myocardium (heart muscle) duringembryogenesis. Patients with mutations causing LVNC develop heartfailure and abnormal cardiac electrophysiology early in life.

Patient specific iPSC-cardiomyocytes (iPSC-CMs) were derived from acohort of LVNC patients as well as family matched control subjects.These cell lines may be used for drug screening and to identifymolecular targets responsible for the disease phenotype.

Methods for the isolation and maintenance of fibroblast cells;derivation of patient-specific iPSC cell lines; and cardiacdifferentiation of cells was performed as described in Example 1 orExample 2.

Example 6 Cardiomyocytes from Patients with DILV

Double Inlet Left Ventricle (DILV). DILV is a congenital heart defect inwhich both the left and right atria feed into the left ventricle. As aresult, children born with this defect only have one functionalventricular chamber, and trouble pumping oxygenated blood into thegeneral circulation.

Patient specific iPSC-cardiomyocytes (iPSC-CMs) were derived from oneindividual with this diease. These cell lines may be used for drugscreening and to identify molecular targets responsible for the diseasephenotype.

Methods for the isolation and maintenance of fibroblast cells;derivation of patient-specific iPSC cell lines; and cardiacdifferentiation of cells was performed as described in Example 1 orExample 2.

Example 7 Cardiomyocytes from Patients with Long QT

Long QT (Type-1) Syndrome (LQT-1, KCNQ1 mutation). Long QT syndrome(LQT) is a hereditary arrhythmic disease in which the QT phase of theelectrocardiogram is prolonged, resulting in increased susceptibilityfor arrhythmia and sudden cardiac death. There are 13 known genesassociated with LQT.

Patient specific iPSC-cardiomyocytes (iPSC-CMs) were derived from acohort of LQT patients carrying a mutation in the KCNQ1 gene, which isthe most commonly mutated LQT gene and responsible for 30-35% of allcases of the disease. The gene had a G269S missense mutation. These celllines may be used for drug screening and to identify molecular targetsresponsible for the disease phenotype.

Methods for the isolation and maintenance of fibroblast cells;derivation of patient-specific iPSC cell lines; and cardiacdifferentiation of cells was performed as described in Example 1 orExample 2.

The preceding merely illustrates the principles of the invention. Itwill be appreciated that those skilled in the art will be able to devisevarious arrangements which, although not explicitly described or shownherein, embody the principles of the invention and are included withinits spirit and scope. Furthermore, all examples and conditional languagerecited herein are principally intended to aid the reader inunderstanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofpresent invention is embodied by the appended claims.

1-32. (canceled)
 33. An in vitro-generated cardiomyocyte wherein: a. thein-vitro-generated cardiomyocyte is generated from a pluripotent stemcell or a reprogrammed cell in vitro; b. the in-vitro-generatedcardiomyocyte comprises at least one mutation in a gene encoding asarcomeric protein; and c. the in-vitro-generated cardiomyocyte displaysa phenotype associated with hypertrophic cardiomyopathy.
 34. The invitro-generated cardiomyocyte of claim 33, wherein thein-vitro-generated cardiomyocyte exhibits, relative to a normalcardiomyocyte, one or more phenotypes selected from the group consistingof: an electrophysiological phenotype, contractile arrhythmia, anincreased intracellular calcium level, and an increased ratio ofβ-myosin expression to α-myosin expression.
 35. The in-vitro-generatedcardiomyocyte of claim 33, wherein the in-vitro-generated cardiomyocyteis generated from the pluripotent stem cell, and the pluripotent stemcell is an induced pluripotent stem cell.
 36. The in-vitro-generatedcardiomyocyte of claim 35, wherein the induced pluripotent stem cell isderived from a subject with hypertrophic cardiomyopathy, arrhythmia, orboth.
 37. The in-vitro-generated cardiomyocyte of claim 33, wherein thegene encoding a sarcomeric protein is cardiac troponin T (TNNT2), myosinheavy chain (MYH7), tropomyosin 1 (TPM1), myosin binding protein C(MYBPC3), 5′-AMP-activated protein kinase subunit gamma-2 (PRKAG2),troponin I type 3 (TNNI3), titin (UN), myosin light chain 2 (MYL2),actin alpha cardiac muscle 1 (ACTC1), or cardiac LIM protein (CSRP3).38. The in-vitro-generated cardiomyocyte of claim 33, further comprisingat least one mutation in caveolin 3 (CAV3), galactosidase alpha (GLA),lysosomal-associated membrane protein 2 (LAMP2), mitochondrial transferRNA glycine (MTTG), mitochondrial transfer RNA isoleucine (MTTI),mitochondrial transfer RNA lysine (MTTK), mitochondrial transfer RNAglutamine (MTTQ), myosin light chain 3 (MYL3), troponin C (TNNC1),Transthyretin (TTR), GATA4, or a combination thereof.
 39. Thein-vitro-generated cardiomyocyte of claim 33, wherein the at least onemutation is in MYH7.
 40. The in-vitro-generated cardiomyocyte of claim33, wherein the at least one mutation is a MYH7 R663H mutation
 41. Thein-vitro-generated cardiomyocyte of claim 33, wherein the phenotypeassociated with hypertrophic cardiomyopathy comprises, relative to anormal cardiomyocyte, electrophysiological arrhythmia, contractilearrhythmia, an increased intracellular calcium level, an increased ratioof β-myosin expression to α-myosin expression, an increased cell size,irregular calcium transient, an increased intracellular calcium level,calcineurin activation, nuclear translocation of nuclear factor ofactivated T-cells (NFAT), or an increased hypertrophic response to apositive inotropic stress.
 42. A population or panel of cells, whereinthe population or panel of cells comprises the in-vitro-generatedcardiomyocyte of claim
 33. 43. The population or panel of cells of claim42, wherein the population or panel of cells has a cardiomyocyte purityof greater than 60%.
 44. A cell culture comprising: a) thein-vitro-generated cardiomyocyte of claim 33; and b) a candidate agentselected from the group consisting of: a calcium channel blocker, asodium channel blocker, a potassium channel blocker, a beta blocker, anda combination thereof.
 45. A method for screening a candidate agent, themethod comprising: a) contacting the candidate agent with acardiomyocyte in vitro, wherein the cardiomyocyte i) comprises at leastone mutation in a gene encoding a sarcomeric protein; and ii) displays aphenotype associated with hypertrophic cardiomyopathy; and b) using anin vitro assay to detect an effect of the candidate agent on thephenotype associated with hypertrophic cardiomyopathy.
 46. The method ofclaim 45, wherein the candidate agent comprises a drug candidate. 47.The method of claim 45, wherein the in vitro assay comprises atomicforce microscopy, microelectrode array recordings, patch clamping,single cell PCR, or calcium imaging.
 48. The method of claim 45, whereinthe cardiomyocyte is generated from a pluripotent stem cell in vitro.49. The method of claim 45, wherein the gene encoding a sarcomericprotein is TNNT2, MYH7, TPM1, MYBPC3, PRKAG2, TNNI3, UN, MYL2, ACTC1,CSRP3, CAV3, GLA, LAMP2, MTTG, MTTI, MTTK, MTTQ, MYL3, TNNC1, TTR, orGATA4.
 50. The method of claim 45, wherein the phenotype associated withhypertrophic cardiomyopathy comprises, relative to a normalcardiomyocyte, electrophysiological arrhythmia, contractile arrhythmia,an increased intracellular calcium level, an increased ratio of β-myosinexpression to α-myosin expression, an increased cell size, irregularcalcium transient, an increased intracellular calcium level, calcineurinactivation, nuclear translocation of NFAT, or an increased hypertrophicresponse to a positive inotropic stress.
 51. The method of claim 45,further comprising subjecting the cardiomyocyte to electricalstimulation.
 52. The method of claim 45, further comprising subjectingthe cardiomyocyte to drug stimulation.
 53. The method of claim 45,further comprising, prior to the contacting the cardiomyocyte with thecandidate agent, generating the cardiomyocyte from a pluripotent stemcell derived from a subject with hypertrophic cardiomyopathy.
 54. Themethod of claim 53, further comprising administering the candidate agentto the subject with hypertrophic cardiomyopathy based on the effect ofthe candidate agent on the phenotype.
 55. The method of claim 45,wherein the candidate agent is a calcium channel blocker or a sodiumchannel blocker.